Biotransformations hydrolase-catalyzed reactions

Based on reactions they catalyze, enzymes can be broadly classified into six major categories (Table 1.1) [1], It was estimated that about 60% of biotransformations currently rely on the use of hydrolases, followed by 20% of oxidoreductases [2]. On the other hand, some of the C—C bond-forming and oxygenation enzymes catalyze reactions with very high reaction efficiency and very low waste generation, underlining the potential of emerging enzymes. 

Hydrolases — especially lipases — proved to be versatile biocatalysts for synthetic biotransformations [79, 80). The vast majority of the enzymatic stereoselective processes have been performed so far in batch mode [29, 30, 81]. Very recently, a review appeared on lipase-catalyzed reactions under continuous-flow conditions [82], and here we extend this overview with an analysis of the range of selectivities, effects of reaction conditions and the mode of enzyme immobilization on the lipase, and in general hydrolase-catalyzed continuous-flow biotransformations. 

Organic aqueous two-phase solvent systems are also widely applied for other biotransformations, for example, in hydrolase-catalyzed processes [37,49]. A typical example is the enantioselective hydrolysis of a racemic, water-immisdble ester as a substrate. The reaction yields a water-soluble carboxylic acid (in depro-tonated form as a salt). Ionic liquids have also been used in biphasic systems, a topic that is discussed in detail in Chapter 3 in this book [50]. 

The metabolism of foreign compounds (xenobiotics) often takes place in two consecutive reactions, classically referred to as phases one and two. Phase I is a functionalization of the lipophilic compound that can be used to attach a conjugate in Phase II. The conjugated product is usually sufficiently water-soluble to be excretable into the urine. The most important biotransformations of Phase I are aromatic and aliphatic hydroxylations catalyzed by cytochromes P450. Other Phase I enzymes are for example epoxide hydrolases or carboxylesterases. Typical Phase II enzymes are UDP-glucuronosyltrans-ferases, sulfotransferases, N-acetyltransferases and methyltransferases e.g. thiopurin S-methyltransferase. 

In this section, enzymes in the EC 2.4. class are presented that catalyze valuable and interesting reactions in the field of polymer chemistry. The Enzyme Commission (EC) classification scheme organizes enzymes according to their biochemical function in living systems. Enzymes can, however, also catalyze the reverse reaction, which is very often used in biocatalytic synthesis. Therefore, newer classification systems were developed based on the three-dimensional structure and function of the enzyme, the property of the enzyme, the biotransformation the enzyme catalyzes etc. [88-93]. The Carbohydrate-Active enZYmes Database (CAZy), which is currently the best database/classification system for carbohydrate-active enzymes uses an amino-acid-sequence-based classification and would classify some of the enzymes presented in the following as hydrolases rather than transferases (e.g. branching enzyme, sucrases, and amylomaltase) [91]. Nevertheless, we present these enzymes here because they are transferases according to the EC classification. 

The principal emphasis of this article is on reactions carried out by isolated enzymes that have the broadest synthetic utility, i.e.. hydrolases, oxidoreductases. and lyases. Biotransforinaliuns catalyzed by living cells are considered to a lesser extent. For more detailed information on biotransformations the reader may consult several books and numerous reviews. 

Some new approaches to suppress competitive reactions in protease-catalyzed peptide synthesis have been developed in our group [14], namely leaving group manipulations at the acyl donor in kinetically controlled reactions, enzymatic synthesis in organic solvent-free microaqueous systems, cryoenzymatic peptide synthesis, and biotransformations in frozen aqueous systems using the reverse hydrolysis potential of proteases and other hydrolases... 

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