Biocatalysis product isolation

In summary, the combination of enzymes is advantageous from an enzymol-ogy and reachon engineering point of view. Reaction yields can be increased by avoiding product inhibition of single enzymatic reachons. Product decomposihon (e.g. by hydrolysis) can be overcome by further enzymatic transformahons. Tedious isolation of intermediate products is not necessary. However, both strategies - combinatorial biocatalysis and combinatorial biosynthesis - have their disadvantages. The in vitro approach needs every enzyme to be produced by recombinant techniques and purified in high amounts, which is in some cases difficult to achieve. On the other hand, product isolation from a biotransformation with permeabilized or whole host cells can be tedious and results in low yields. 

Enantioselective catalysis (and kinetic resolution) where the expensive chiral auxiliary is used in substoichiometric amounts attached to an active catalytic entity. The catalysts can be man-made (chemical catalysis), enzymes or whole cells (biocatalysis). In many respects, both chemical and biocatalysts have similar problems when used on an industrial scale (for chemical catalysts, see below). In biocatalysis, the often high effort for finding and developing an efficient biocatalyst, especially when the starting material is not a very close analog to the natural substrate, and the product isolation from an often rather dilute aqueous solution can be problematic. 

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... 

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. 

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. 

Despite the limitations, the great success of enzyme immobilization in diagnostics, pharmaceutical, food and chemical industries is undeniable12, 3. The decision whether one should use a soluble enzyme preparation or an immobilized enzyme does not have a universal solution and can be decided only on a case by case basis. Ordinarily, if the cost of an enzyme represents a significant portion of the overall cost or if isolation of the final product is complicated by the presence of the soluble protein, the cost of immobilization can be offset by the gains in productivity and improved product quality. The intent of this section is to describe, in general terms with illustrative examples, the features and considerations of these broad classes of enzyme immobilization as they impact their application to biocatalysis. Detailed experimental protocols are available in the original literature and exemplary protocols for these methods are offered in many excellent reviews and texts 14L... 

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. 

All biotechnological processes depend on enzymes - either in whole-cell living systems or isolated out of their biological context. In fact, purified enzymes are established in processing food and textiles and are supplements in feed and detergents - all products of everybody s daily life. Whole-cell systems particularly in the field of specialty chemicals provide a broad range of methods and processes. For many years, enantiomerically pure amino acids for food, feed and pharma industries have been produced by microbial fermentation. Ambitious R D resulted in enzymes especially modified and optimized for a desired chemical reaction such as biocatalysis of optically active amines, alcohols, epoxides and more. ... 

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