Enzymes drawbacks

As shown in this chapter, by focusing on the modulation of enzyme selectivity by medium engineering, quite simple modifications of the solvent composition can really have significant effects on the performances of the biocatalysts. The main drawback remains the lack of reliable predictive models. Despite the significant research efforts (particularly in the last decade), it is likely that a reasonable foresight of the enantioselective outcome of an enzymatic transformation will continue to be based solely on a careful analysis of the increasingly numerous literature reports. 

Compared to synthetic catalysts, enzymes have many advantages. First of all, being natural products, they are environmentally benign and therefore their use does not meet pubhc opposition. Enzymes act at atmospheric pressure, ambient temperature, and at pH between 4 and 9, thus avoiding extreme conditions, which might result in undesired side reactions. Enzymes are extremely selective (see below). There are also, of course, some drawbacks of biocatalysts. For example, enzymes are known in only one enantiomeric form, as they consist of natural enantiomeric (homochiral) amino acids their possible modifications are difficult to achieve (see Section 5.3.2) they are prone to deactivation owing to inappropriate operation parameters and to inhibition phenomena. 

For detailed description and discussion of methods of separation and characterization of GAG, the reader is referred to specific mono-graphs38-42-46-47 dealing with the advantages and drawbacks of different colorimetric, titrimetric, electrophoretic, chromatographic, spectroscopic, and enzymic methods for the qualitative and quantitative characterization of heparin and its most common contaminants. The present article is concerned only with analytical aspects of relevance to the structural characterization of heparin. 

An alternative to chemical synthesis is to use human CYP enzymes to generate the desired human drug metabolites. Various means of making human P450s have been used, all with certain drawbacks [81]. The most common source is pooled HLMs, which has been described in detail previously, but these microsomal preparations contain a mixture of many different enzymes, and their cost, batch-to-batch variability in activity and restrictions on availability can limit the usefulness of HLMs for preparative synthetic work. These limitations can become particularly acute when the required amount of a pure metabolite exceeds 5-10 mg. 

The improvement of its activity and stability has been approach by the use of GE tools (see Refs. [398] and [399], respectively). A process drawback is the fact that the oxidation of hydrophobic compounds in an organic solvent becomes limited by substrate partition between the active site of the enzyme and the bulk solvent [398], To provide the biocatalyst soluble with a hydrophobic active site access, keeping its solubility in organic solvents, a double chemical modification on horse heart cytochrome c has been performed [400,401], First, to increase the active-site hydrophobicity, a methyl esterification on the heme propionates was performed. Then, polyethylene glycol (PEG) was used for a surface modification of the protein, yielding a protein-polymer conjugates that are soluble in organic solvents. 

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