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Biotransformations are those reactions brought about by the application o f biological catalysts. The biocatalyst, may either be free in t ^ reaction mixture, immobilised, or within a whole cell system, which again may be mobile or attached to a support.

1.2.1 Enzymes

There are several advantages and disadvantages to using enzymes to effect a reaction rather than enq>loying chemical means, and a few o f these are briefly mentioned below. Faber, (1992) and Poppe & Novak, (1992) provide a comprehensive and well conçiled list o f the benefits and problems encountered with the use o f biocatalysts.

• Enzymes have the abihty to catalyse a number o f different classes o f reaction. They may be used as an alternative to chemical methods, or may be used to perform those reactions that cannot be brought about using chemical means.

• The source o f activation energy with the use o f enzymes is the conformational

change in the proteins’ structures. With other processes, this activation energy must be provided, usually as thermal energy.

• Enzymes fimction optimally under nnld reaction conditions. These moderate

environments are easy to create and increase the stabihties o f labile conq)onents involved in the reaction. The use o f toxic, highly reactive chemicals is eliminated, thus decreasing the risk environmental damage.

• Those conditions that affect enzyme activity and selectivity, e.g. temperature,

pH and reactant/product concentrations, can be manipulated in order to achieve a desired effect in the biotransformation.

• Enzyme catalysed reactions are selective with regard to the substrates used and

the type o f reaction catalysed. Some biocatalysts are capable o f specifically producmg optically active molecules in an enantiomericaUy pure form. This can allow resolution o f racemic mixtures o f substrates or can result m asymmetric syntheses.

There are also disadvantages associated with the use o f enzymes:

• Although there are a large number o f enzymes that have been characterised,

relatively few are commercially available in either fi:ee or immobilised form.

• Due to their biochemical origins, enzymes do not fimction well in conditions

away fi*om their optima; for exan^le, enzymes usually work best in aqueous environments, although this may not be the ideal medium for the other reaction components.

• Enzymes are usually sensitive to certain concentrations o f reactants and

products, which can limit the amounts present in a reactor, by being toxic or inhibitory. The types o f inhibition possible in an enzyme catalysed reaction are

shown in Figure 1.1. Different forms o f product inhibition are represented in Figure

1.2.

• Pure enzymes are expensive, and some enzymes are unstable.

• Some enzymes are highly specific with regard to the substrates they use. An

ideal enzyme would be able to utilise a wide range o f substrates, whilst maintaining a high degree o f stereoselectivity.

1

r

It

-►B

Figure 1.1 Inhibition effects within a biotransformation: S = substrate, P = product, B = by-product; (1) product inhibition, (2) feedback inhibition, (3) substrate inhibition, (4 & 5) unspecific inhibition by side-products (obtained from Mattiasson & Holst, 1991).

No product inhibition (Michaelis-Menten kinetics) _ Non-specific ; deactivation Competitive product inhibition Non-competitive product inhibition

(S]

Although there are drawbacks associated with the use o f biocatalysts in biotransformations, most o f these can be alleviated in one way or another. Enzymes may be expensive and some^\iiat labile, but there are methods o f inçroving their stability. Genetic modification, removal o f proteolytic agents, immobilisation and the use o f stabilisers all contribute toward bringing the costs o f biocatalysts down by increasing their stability. Likewise, the use o f clarified enzyme preparations are cheaper than using purified enzyme. Hence, the special properties o f enzymes, e.g. their cbemoselectivity, regioselectivity, diastereoselectivity, enantioselectivity and the fact that they are chiral catalysts can be used to perform biotransformations whilst retaining stabihties.

1.2.2 Examples and applications

There are numerous enzymatic reactions that can be executed to perform useful organic chemistry. Broadly, they can be classified into the following types: oxidoreductases, transferases, hydrolases, lyases, isomerases and hgases.

-Again, Faber, (1992) and Poppe & Novak, (1992) provide in-depth reviews o f

well-estabhsbed biotransformations. More recent reviews o f the latest

developments in biotransformations are provided by Roberts & Turner, (1992) and

Roberts et ah, (1995). A summary o f those biotransformations performed on an

industrial scale are covered by Kieshcb, (1991) and Lihy, (1992).

1.2.3 Biotransformation process design

The design o f a biotransformation involves the integration and development o f various aspects o f molecular biology, reactor design and operation, fermentation and downstream operation methodologies, such that there is a maximal quantity o f product formed with minimal expense, time and effort.

• The correct choice o f biocatalyst must be made; it must perform the desired bioconversion with relative ease, for example, such that its activity is not easily decreased with slight changes in temperature.

• A good biocatalyst production system should be available. This normally

involves using a recombinant host organism containing a plasmid vector holding genetic information necessary for the expression o f the desired enzyme. This recombinant host must be easy to grow on a large scale, possess a means o f identifying recombinant cells from non-recombinants, maintain high levels o f plasmid retention and have a straightforward approach to obtaining the enzyme in a form that allows it to be used for the biotransformation.

• There are several factors that need consideration in the conq)letion and

inq)lementation o f the actual biotransformation reaction. ExaiDples are catalyst preparation, reactor selection and reactor kinetics.

• Finally the recovery o f the product from the bioreaction mixture requires

consideration. The information needed in choosing a relevant downstream processing protocol will discussed in the following section. Reviews into the criteria for the correct selection o f design and operation parameters o f biotransformation processes are provided by Lilfy, (1992), Lilly & Woodley, (1996) and Woodley & Lilly, (1994).