Chapter 1

Introduction, Types of Reactions, and Sources of Biocatalysts

Animesh Goswami* Jon D. Stewart┼
*    Chemical Development, Bristol-Myers Squibb, New Brunswick, NJ, USA
┼    Department of Chemistry, University of Florida, Gainesville, FL, USA

Abstract

This chapter defines the scope and limits of the book along with its focus on using enzymes for preparative synthesis. Enzymes are highly selective catalysts, and their contributions to chemo-, regio-, and stereoselectivities are described. Commonly required cofactors are briefly described along with relevant aspects of enzyme kinetics. A brief survey of common reaction types in biocatalysis covers both the reaction and associated enzyme types; a comprehensive table is provided as well. The various sources of biocatalysts (animals, plants, and microorganisms) are discussed along with their relative advantages and disadvantages. The uses of soluble versus insoluble enzyme forms are introduced and some benefits of each strategy are cited.

Keywords

biocatalysis enzyme chemoselectivity regioselectivity enantioselectivity enzyme kinetics cofactors common biocatalytic reactions sources of biocatalysts enzyme forms

1. Introduction

1.1. Enzymes and Their Roles in Nature

Enzymes are nature's catalysts, facilitating the creation, functioning, maintenance, and ultimately the demise of all living cells. Enzymes are proteins composed of 20 natural amino acids joined together by peptide bonds, in some cases augmented with additional organic or inorganic species known as cofactors.1 In addition to their primary molecular structures dictated by the amino acid sequence, enzyme catalytic function also depends upon subsequent folding into specific three-dimensional shapes that contain a variety of secondary and tertiary structural elements. These architectures determine not only enzyme function but also how they interact with the external solvent medium in which they are dissolved or suspended. This can have important ramifications when enzymes are employed under partially or completely nonaqueous conditions. Like all catalysts, enzymes increase reaction rates by lowering their activation energies. The most important difference between enzymes and simple catalysts such as a proton or hydroxide is that the former are much more restrictive in the range of acceptable substrates. The three dimensional structure of the enzyme allows binding of only those starting materials (usually referred to as substrates) whose structures are congruent with the size, shape and polarity of the catalytic portion of the enzyme (the "active site"). Formation of this noncovalent complex prior to chemical conversion is the key to the high selectivity of enzyme-catalyzed reactions since it places the substrate into a specific location where its functional groups are oriented precisely with those on the enzyme. Noncovalent complex formation allows chemical reactions between specific amino acids on the enzyme and functional groups on the substrate to occur in an environment that is kinetically equivalent to a unimolecular process. Transforming what would otherwise be bimolecular reactions into effectively intramolecular conversions is a major reason that enzymes can accelerate reactions by up to 23 orders of magnitude over the background (uncatalyzed) reaction [1]. Selectivity is the second benefit from forming a noncovalent enzyme-substrate complex prior to chemical conversion. By focusing the catalytic attention of the enzyme onto a specific area of the substrate, reactions can be restricted to a single portion of the molecule that may or may not be the most reactive portion of the overall substrate structure. This allows enzyme-catalyzed reactions to be selective in many respects: chemoselective (carrying out only one specific transformation while others are possible), regioselective (transforming only one among several possible sites), and stereoselective (producing and/or consuming one stereoisomer in preference to others).

2. Definition of biocatalysis

In nature, enzymes catalyze transformations of metabolites that occur within and/or outside of living cells. Although some enzymes accept only a limited variety of substrates, a large fraction is more tolerant and allows conversions of nonnatural starting materials. The field of biocatalysis rests upon this partial promiscuity. If enzymes were truly selective for only a single substrate, it would be impossible to utilize them for synthesizing new molecules from nonnatural substrates. The goal is to identify or engineer enzymes that are sufficiently general to accept a variety of related substrates, but selective enough to yield single products or stereoisomers. We use the term "biocatalysis" to describe the use of enzymes (either native or modified) for synthetic transformations of nonnatural starting materials. Enzymes used for in vitro synthetic transformations are called biocatalysts, and the processes are called biocatalytic transformations.

3. Scope of this book

Some enzymes catalyze the reactions that build up large molecules from simple building blocks, for example, complex carbohydrates from carbon dioxide and water or the synthesis of steroids and terpenoids from acetate. Others are involved in the degradation of large assemblies to small molecules, for example, hydrolysis of proteins to amino acids and the oxidative degradation of lignin. Although some of these native conversions are industrially important and practiced on large scales, the use of enzymes to produce their normal primary and secondary products of cells lies outside the scope of this book. Here, our focus is on preparing nonnatural compounds using enzymes since this addresses the need commonly encountered in organic synthesis. However, it should be noted that the native reactions of an enzyme can often be used as starting points for their applications to nonnative reactions. The field of metabolic engineering also lies outside the scope of this book. These efforts use two or more enzymes to catalyze sequential steps in a pathway that links a simpler starting material such as glucose with a final intracellular target product such as butanol or lysine. In some cases, the complete pathway already exists within a single organism; in others, enzymes from different sources are assembled into an artificial metabolic pathway in a suitable host cell. The key difference between biocatalysis and metabolic engineering is that the molecular skeletons are provided in vitro in the former case and in vivo in the latter. Although it is economically attractive to produce a target molecule by metabolic engineering, this benefit must be balanced against the (usually) lengthy optimization phase required for efficient production and the restriction that intermediates and the final product should be nontoxic to the host cells.

4. Key benefits of employing enzymes in synthesis

Enzymes offer several attractive features as catalysts for organic synthesis. They often show high selectivities, they can operate under mild conditions and they are completely biodegradable catalysts constructed solely from renewable resources. They are thus ideal strategies as chemistry embraces sustainability.

4.1. Selectivity: Chemo-, Regio- and Stereo-

Biocatalytic reactions can show very high selectivities in all respects (Figure 1.1). When similarly-reactive functional groups are present in a molecule, the enzymes often catalyze only the reaction of one while leaving the others intact. For example, nitrile hydratases catalyze the partial hydrolysis of a nitrile group to yield a primary amide without cleaving an ester moiety present in the same molecule or further hydrolyzing the amide product, a property referred to as "chemoselectivity." Figure 1.1 Types of selectivity exhibited by enzymes.
Biocatalytic processes can provide chemo-, regio-, and stereoselective conversions. Representative examples can be observed in reactions catalyzed by nitrile hydratases, lipases and dehydrogenases. "Regioselectivity" is another useful property displayed by many enzymes. This refers to the transformation of one functional group while leaving other identical (or nearly identical) moieties at different locations within the molecule untouched. For example, among three esters in a triacylglyeride, many lipases hydrolyze only one position, and do not catalyze further hydrolysis of the diester product. All but one of the amino-acid building blocks have at least one chiral center,2 and for this reason, enzymes are intrinsically asymmetric catalysts. The asymmetric nature of enzymes results in enantioselectivity when biocatalysts convert a prochiral starting material into a single product enantiomer, for example, in ketone or imine reductions. The same asymmetric nature also causes the biocatalysts to preferentially transform only one stereoisomer of a starting material that contains a mixture of diastereomers or enantiomers. Such a process is referred to as a kinetic resolution and the ratio of rate constants for the fast- and slow reacting enantiomers is termed the...