Carboxylic acid hydrolase

Of special interest is one group of carboxylic acid hydrolases the lipases (EC 3.1.1.3). Lipases catalyse the hydrolysis of triglycerides into diglycerides, monoglycerides, glycerol and fatty acids. Some lipases are also capable of hydrolysing polyesters to monomeric or oligomeric products which can be taken up by microbial cells and metabolised further by other esterases [18]. 

Before discussmg the mechanism of cleavage of carboxylic acid esters and amides by hydrolases, some chemical principles are worth recalling. The chemical hydrolysis of carboxylic acid derivatives can be catalyzed by acid or base, and, in both cases, the mechanisms involve addition-elimination via a tetrahedral intermediate. A general scheme of ester and amide hydrolysis is presented in Fig. 3. / the chemical mechanisms of ester hydrolysis will be... 

The chemical diversity of carboxylic acid esters (R-CO-O-R ) originates in both moieties, i.e., the acyl group (R-CO-) and the alkoxy or aryloxy group (-OR7). Thus, the acyl group can be made up of aliphatic or aromatic carboxylic acids, carbamic acids, or carbonic acids, and the -OR7 moiety may be derived from an alcohol, an enol, or a phenol. When a thiol is involved, a thioester R-CO-S-R7 is formed. The model substrates to be discussed in Sect. 7.3 will, thus, be classified according to the chemical nature of the -OR7 (or -SR7) moiety, i.e., the alcohol, phenol, or thiol that is the first product to be released during the hydrolase-catalyzed reaction (see Chapt. 3). Diesters represent substrates of special interest and will be presented separately. 

One of the most actively investigated aspects of the biohydrolysis of carboxylic acid esters is enantioselectivity (for a definition of the various stereochemical terms used here, see [7], particularly its Sect. 1.5) for two reasons, one practical (preparation of pure enantiomers for various applications) and one fundamental (investigations on the structure and function of hydrolases). The synthetic and preparative aspects of enantioselective biocatalysis by hydrolases have been extensively investigated for biotechnology applications but are of only secondary interest in our context (e.g., [16-18], see Sect. 7.3.5). In contrast, the fundamental aspects of enantioselectivity in particular and of structure-metabolism relationships in general are central to our approach and are illustrated here with a number of selected examples. 

The preferred route for reducing the molecular weight of PVA involves chain scission at the 1,3-diketone site (see Fig. 6). As the diketone element is chemically not very stable, a spontaneous degradation of oxidised PVA was also discussed [80]. Nevertheless, the preferred degradation pathway is most likely the biochemical process because enzymes were identified that showed high activity with diketone substrates [81], especially with oxidised PVA. The p-diketone hydrolase (BDH EC 3.7.1.7) hydrolyses aliphatic p-diketones to form methyl ketones and carboxylic acids in equimolar amounts [82]. The enzymatic cleavage of C-C bonds in p-diketones is not well studied [83]. BDH enzymes could be isolated from different PVA-degrading strains, purified, characterised and cloned [84]. 

In addition to the now well-documented lipase system, cows milk contains several other carboxyl ester hydrolases, collectively referred to as esterases. These are distinguished from lipases by their ability to act on ester substrates in solution rather than in an emulsified form (Jaeger et al., 1994) and/or by their preference for hydrolysing esters of short-chain rather than long-chain acids (Okuda and Fujii, 1968). 

It is well known that organophosphates, carbamates and sulfonates are acid-transfer-inhibitors of serine hydrolases because they transfer the acid moiety of the inhibitor to the serine hydroxyl of the enzyme active site (34). Extensive evidence indicates that the reaction of these inhibitors with acetylcholinesterases (AChE) appears to involve the same reaction pathway as that for the esters of carboxylic acids, i.e. acetylcholine (see (35) for review), and in fact these inhibitors are considered to be poor substrates of AChE (36), especially the carbamic acid esters ("Equation 30 ). 

Nitrilases and amidases belong to the class of hydrolases and nitrile hydratase belongs to the class of lyase. Nitrilases are an important class of nitrilase superfamily that convert nitrile to the corresponding carboxylic acids and ammonia, whereas nitrile hydratase first converts into the corresponding amide and then this amide is transformed by amidase. There are very few reports for the surface modification of PAN and PA for increasing its hydrophilicity using nitrilases, nitrile hydratases, and amidases. 

The PARKS-linked PD gene has been reported as the ubiquitin carboxyl-terminal hydrolase-Ll (UCHLl) located on chromosome 4pl3 (Leroy et al., 1998). UCHLl is a protein of 223 amino acids, 9 exons, and a transcript length of 1172 bps (Wilkinson et al., 1989) (Fig. 8). 

Pig Liver Esterase (PLE). This is the more used car-boxylesterase (carboxylic-ester hydrolase, EC 3.1.1.1, CAS 9016-18-6) which physiologically catalyzes the hydrolysis of carboxylic acid esters to the free acid anion and alcohol. PLE is a serine hydrolase which has been widely used for the preparation of chiral synthons and these applications have been fully reviewed. An active-site model for interpreting and predicting the specificity of the enzyme has been published. In the pioneering studies of the enzyme applications field, PLE was used for the chiral synthesis of mevalonolactone. Prochiral 3-substituted glutaric acid diesters... 

Hydrolase Ester hydrolysis Alcohol/carboxylic acid/carboxylic ester... 

Amides can in most cases be readily synthesized from activated derivatives of carboxylic acids and amines. They are fairly stable and often need harsh conditions for their removal. On the one hand this prompted a search for advanced methods for their selective cleavage on the other, however, amides generally are used for the protection of chemically stable compounds, e.g. in nucleotide chemistry. In more recent developments, acceleration of the deprotection reaction by intramolecular attack and the advantageous properties of amido hydrolases have been exploited. 

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