Biocatalysis products

For the successful coinmerciahsation of almost all biocatalysis products, patent protection is essential since without patent protection, and the prospect of a period of enforceable monopoly in which to recoup investment and make a profit, it is unlikely that the substantial investment required for bringing a product process to the market will occur. Frequently, the compare making the invention does not wish to, or is not able to, bring the invention to the market itself. Instead, it enters into an alhance with a partner which is better able to do this. In this case, and especially in the period where it is still uncertain that the product will be useful or commercially successful, patents and patent applications, and the know-how associated with the product or processes, may be the most valuable asset of the company. 

Rates of enzyme turnovers can be extremely high in favorable cases, so that only a very small amount of biocatalyst can supply a large amount of biocatalysis product. 

The term biotransformation or biocatalysis is used for processes in which a starting material (precursor) is converted into the desired product in just one step. This can be done by use either of whole cells or of (partially) purified enzymes. Product examples range from bulk chemicals (such as acrylamide) to fine chemicals and chiral synthons (chiral amines or alcohols, for example). There are several books and reviews dealing with the use of bio transformations either at laboratory or at industrial scales [1, 10-13]. 

Since stereoselectivities of biocatalytic reductions are not always satisfactory, modification of biocatalysis are necessary for practical use. This section explains how to find, prepare, and modify the suitable biocatalysts, how to recycle the coenzyme, and how to improve productivity and enantioselectivity of the reactions. 

Recently, recombinant biocatalysts obtained using Escherichia coli cells were designed for this process. The overexpression of all enzymes required for the process, namely, hydantoinase, carbamoylase, and hydantoin racemase from Arthrobacter sp. DSM 9771 was achieved. These cells were used for production of a-amino acids at the concentration of above 50 g 1 dry cell weight [37]. This is an excellent example presenting the power of biocatalysis with respect to classical catalysis, since a simultaneous use of three different biocatalysts originated from one microorganism can be easily achieved. 

However, the reactions were not enantioselective ones, though the most important aspect of the biocatalysis reaction should be in the enantioselective reaction. We and KragF independently reported the first enantioselective lipase-catalyzed reaction in February-March 2001. Since lipase was anchored by the IL solvent and remained in it after the extraction work-up of the product, we succeeded in demonstrating that recyclable use of the lipase in the [bmim][PFg] solvent system was possible (Fig. 2). ... 

Another advantage of biocatalysis is that chemo-, regio-, and stereoselectivities are attainable that are difficult or impossible to achieve by chemical means. A pertinent example is the production of the artificial sweetener, aspartame, which has become somewhat of an industrial commodity. The enzymatic process (Fig. 2.31), operated by the Holland Sweetener Company (a joint venture of DSM and Tosoh), is completely regio- and enantiospecific (Oyama, 1992). 

Bruggink (1996) has given an account of how the production of cefalexin, which is the largest cephalosporin in the market, can be converted from a ten-step process based on benzaldehyde and penicillin into a six-step process where biocatalysis is involved in three steps. The wastewater stream, containing 30-40 kg of unwanted materials in the conventional process, has been substantially reduced. Similarly, Van Loon et al. (1996) have given details of fermentation processes for cleaner and cheaper compared to the process practised so far. 

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... 

There are many advantages of two-phase systems over aqueous systems when they are used as biocatalysis media [21,22,26,27] components (substrates and/or products) can be... 

Biocatalysis localization in the biphasic medium depends on physicochemical properties of the reactants. When all the chemical species involved in the reaction are hydro-phobic, catalysis occurs at the liquid-liquid interface. However, when the substrate is hydrophobic (initially dissolved in the apolar phase) and the product is hydrophilic (remains in the aqueous phase), the reaction occurs in the aqueous phase [25]. The majority of biphasic systems use sparingly water-soluble substrates and yield hydrophobic products therefore, the aqueous phase serves as a biocatalyst container [34,35] [Fig. 2(a)]. Nevertheless, in some systems, one of the reactants (substrate or product) can be soluble in the aqueous phase [23,36-38] (Fig. 2(b), (c)). 

A common characteristic of metabolic pathways is that the product of one enzyme in sequence is the substrate for the next enzyme and so forth. In vivo, biocatalysis takes place in compartmentalized cellular structure as highly organized particle and membrane systems. This allows control of enzyme-catalyzed reactions. Several multienzyme systems have been studied by many researchers. They consist essentially of membrane- [104] and matrix- [105,106] bound enzymes or coupled enzymes in low water media [107]. 

The present section deals with the improvement in the performance of biocatalysis when carried out in organic-aqueous biphasic systems. Such systems are very useful in equilibrium reactions and conversion yield where substrates and products can be dissolved and drawn into different phases. Subsequently the synthesis in the reactive aqueous phase is allowed to continue. 

The stirred batch reactors are easy to operate and their configurations avoid temperature and concentration gradient (Table 5). These reactors are useful for hydrolysis reactions proceeding very slowly. After the end of the batch reaction, separation of the powdered enzyme support and the product from the reaction mixture can be accomplished by a simple centrifugation and/or filtration. Roffler et al. [114] investigated two-phase biocatalysis and described stirred-tank reactor coupled to a settler for extraction of product with direct solvent addition. This basic experimental setup can lead to a rather stable emulsion that needs a long settling time. 

Green Chemistry with Biocatalysis for Production of Pharmaceuticals 305... 

The cholesterol-lowering drug atorvastatin, marketed as Lipitor, is an example where biocatalysis research has been applied extensively and is in industrial use. The enzyme 2-deoxyribose-5-phosphate aldolase (DERA) has been a target of directed evolution for the production of atorvastatin intermediates [8,9,71]. DeSantis and coworkers [8,9] used structure-based... 

The complexity of today s pharmaceutical compounds and an increasing awareness of the environmental impact of traditional chemical syntheses have opened the door to biocatalysis. Directed evolution is an integral tool in the development of synthetic enzymes, ensuring they are suitable for use in an industrial setting. The past success of this approach indicates that it will continue to provide many examples of safe and efficient production of chemical intermediates and medical compounds. 

Itoh, N., Nakamura, M., Inoue, K. and Makino, Y. (2007) Continuous production of chiral 1,3-butanediol using immobilized biocatalysts in a packed bed reactor promising biocatalysis method with an asymmetric hydrogen-transfer bioreduction. Applied Microbiology and Biotechnology, 75 (6), 1249-1256. 

Cheetham, P.S.J. (1994) Case studies in applied biocatalysis - from ideas to products, in Applied Biocatalysis (eds J.M.S. Cabral, D. Best, L. Boross and J. Tramper), Harwood Academic Publishers, Chur, Switzerland, pp. 87-89. 

Hamann, M.T. (2003) Enhancing marine natural product structural diversity and bioactivity through semisynthesis and biocatalysis. Current Pharmaceutical Design, 9, (11) 879-889. 

It has also been recognized and it is widely accepted that biocatalysis can make a difference and has a strong impact on many principles of green chemistry. This impact goes much beyond pharmaceuticals production, which, however, will be the focus of this chapter. 

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