Organic enzyme properties

Biocatalysis refers to catalysis by enzymes. The enzyme may be introduced into the reaction in a purified isolated form or as a whole-cell micro-organism. Enzymes are highly complex proteins, typically made up of 100 to 400 amino acid units. The catalytic properties of an enzyme depend on the actual sequence of amino acids, which also determines its three-dimensional structure. In this respect the location of cysteine groups is particularly important since these form stable disulfide linkages, which hold the structure in place. This three-dimensional structure, whilst not directly involved in the catalysis, plays an important role by holding the active site or sites on the enzyme in the correct orientation to act as a catalyst. Some important aspects of enzyme catalysis, relevant to green chemistry, are summarized in Table 4.3. 

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. 

Further advantages of biocatalysis over chemical catalysis include shorter synthesis routes and milder reaction conditions. Enzymatic reactions are not confined to in vivo systems - many enzymes are also available as isolated compounds which catalyze reactions in water and even in organic solvents [28]. Despite these advantages, the activity and stability of most wild-type enzymes do not meet the demands of industrial processes. Fortunately, modern protein engineering methods can be used to change enzyme properties and optimize desired characteristics. In Chapter 5 we will outline these optimization methods, including site-directed mutagenesis and directed evolution. 

Enzymes are powerful biological catalysts that are essential for the proper maintenance and propagation of any organism. These properties make them... 

Biological systems are, by definition, multicomponent systems. One should keep in mind the difficulties of constructing molecular level pictures that satisfactorily describe systems such as a protein in a reverse micelle or a protein in a concentrated aqueous salt solution, which are certainly much simpler than anhydrobiotic organisms, for example. It is not clear to what extent the water of the hydration shell can be replaced by a third component (e.g., lipid) or what effect such replacement has on protein or enzyme properties. 

Campanella, L. Favero, G. Sammartino, M. P. Tomassetti, M., Further development of catalase, tyrosinase and glucose oxidase based organic phase enzyme electrode response as a function of organic solvent properties, Talanta 1998, 46, 595-606... 

Colombo, G., Ottolina, G. and Carrea, G., Modelling of enzyme properties in organic solvents, Monatshefte fuer Chemie, 2000, 131, 527-547. 

With directed evolution we can engineer enzyme properties rapidly and with a high probability of success. Many enzymes that have been improved by directed evolution are listed in Tab. 4-3. This powerful biocatalyst engineering strategy creates new opportunities in organic synthesis new and improved bioconversion processes can be developed and novel compounds that are otherwise inaccessible by classical chemistry can be synthesized. In addition, the molecules created by directed evolution offer an excellent opportunity for improving our still poor understanding of sequence-structure-function relationships. 

Several lines of evidence indicate that neoplastic cells per se are the main source of extracellular thiol proteinase activity. Recent studies (39) have shown that malignant human breast tumors maintained in organ culture secrete high levels of a cathepsin B-like enzyme into the culture medium. Moreover high levels of cathepsin B-like enzyme are present in the serum of patients with a wide variety of cancers, and these levels decrease when the cancer tissue is removed or treated with therapeutic agents (64, 65). Cathepsin B-like enzyme from cultured cells of malignant tumors (39,66) possesses enzymic properties similar to those of cathepsin B with respect to specificity, affinity, and pH optima for synthetic substrates. It hydrolyzes Bz-Arg-Arg-2-naphthylamide and is inhibited by leupeptin. However, the tumor enzyme is much more stable than cathepsin B to inactivation above pH 7. It has a molecular weight of about 33,000-35,000. The distribution of cathepsin B-like activity was determined in fractions of control and neoplastic epithelial cells from human ectocervix (66). The activity is present mainly in the mitochondrial and lysosomal fractions of normal cells but mainly in the plasma membranes and nuclei of neoplastic cells. 

The affinity labeling reagents described above have proved to be useful in studies of the active sites of lysozymes isolated from different organisms. Many of these lysozymes exhibit close similarity in their amino acid sequences, fluorescence spectra, and enzymic properties including not only the ability to digest cell walls of M. luteus, but also to catalyze transglycosylation reactions with a variety of saccharide acceptors when the cell wall tetrasaccharide or chitin oligosaccharides are used as substrates. ... 

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