Biocatalysis hydrolases

In biocatalysis, hydrolases are the most important class of enzymes for carrying out enzymatic resolutions. Many hydrolases, such as esterases, lipases, epoxide hydrolases, proteases, peptidases, acylases, and amidases, are commercially available a substantial number of them are bulk enzymes [87]. Resting-cell systems, if they are not immobilized, are used in diluted suspensions and could be handled as quasi-homogeneous catalysts. 

In the last decade, biocatalysis in nonaqueous media, using hydrolases, has been widely used for organic chemists. The possibilities that these biocatalysts offer for the preparation of different types of organic compounds, depending upon the nucleophile... 

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. 

Biocatalysis is still an emerging field hence, some transformations are more established than others.Panke et alP have performed a survey of patent applications in the area of biocatalysis granted between the years 2000 and 2004. They found that although hydrolases, which perform hydrolyses and esterifications, still command widespread attention and remain the most utilized class of enzyme (Figure 1.5), significant focus has turned towards the use of biocatalysts with different activities and in particular alcohol dehydrogenases (ADHs) - also known as ketoreductases (KREDs) - used for asymmetric ketone reduction. 

In addition to the retention of structural integrity in neat organic solvents, Klibanov and co-workers demonstrated that a diverse range of enzymes, from hydrolases and peroxidases to cofactor-dependent alcohol oxidases and ADHs, also retain activity. This pioneering work single-handedly led to the popularization of biocatalysis in neat organic solvent. 

Keywords Epoxide, Vicinal diol. Epoxide hydrolase. Biocatalysis, Enantio-convergent. 

Whereas several areas of biocatalysis - in particular the use of easy-to-use hydrolases, such as proteases, esterases and lipases - are sufficiently well research to be applied in every standard laboratory, other types of enzymes are still waiting to be discovered with respect to their applicability in organic-chemistry transformations on a preparative scale. This latter point is stressed in this volume, which concentrates on the newcomer-enzymes which show great synthetic potential. 

Originally almost all apphcations of biocatalysis involved hydrolytic reactions, except a few, such as L-sorbose and ephedrine manufacture (Turner, 1994). Hydrolases still are the main conunercial enzyme class, but nowadays a much wider range of reactions is being applied, either on a conunercial scale or on a lab scale. The most important reaction types are reviewed in chapter 2. 

Faber, K., Ottolina, G. andRiva, S. (1993) Selectivity-Enhancement of Hydrolase Reactions. Biocatalysis, 8, 91-132. 

Enzymes that are suited for application in biocatalysis are mostly hydrolases, bnt also oxidorednctases, lyases and, to a lesser extent, transferases are useful. Obviously, the focus of bulk enzyme producers is different from the main interests of those who want to apply enzymes in biocatalytic applications. Fortunately, a growing number of companies has become active in the field of enzyme prodnction for biotransformations and by now a large nnmber of enzymes suited for biotransformations has become commercially available (Table 5.1). 

HNLs comprise a heterogenous enzyme family, since hydroxynitrile lyase activity has evolved in different structural frames by convergent evolution [17, 18]. Thus, (S) -specific HNLs based on an a/P-hydrolase fold framework from Manihot esculmta (cassava) [19-21], Hevea hrasilensis (rubber tree) [22-26], and Sorghum hicolor (millet) [27-33] have been described. (R)-specific HNLs based on the structural framework of oxidoreductases were isolated from Linum usitatissimum (flax) [30, 34-37] and Rosaceae (e.g., bitter almonds) [31, 38]. Despite their potential in biocatalysis only few HNLs (from cassava and rubber tree) are available by recombinant gene expression, which is a prerequisite for their technical application [20, 24]. Thus, cloning, recombinant expression, and... 

Microreactor technology offers the possibility to combine synthesis and analysis on one microfluidic chip. A combination of enantioselective biocatalysis and on-chip analysis has recently been reported by Beider et al. [424]. The combination of very fast separations (<1 s) of enantiomers using microchip electrophoresis with enantioselective catalysis allows high-throughput screening of enantioselective catalysts. Various epoxide-hydrolase mutants were screened for the hydrolysis of a specific epoxide to the diol product with direct on-chip analysis of the enantiomeric excess (Scheme 4.112). 

Chiappe, C., Leandri, E., Lucchesi, S., Pieraccini, D., Hammock, B.D., and Morisseau, C. 2004. Biocatalysis in ionic liquids The stereoconvergent hydrolysis of trans- -methylstyrene oxide catalyzed by soluble epoxide hydrolase. Journal of Molecular Catalysis B Enzymatic, 27 243 8. 

It is generally stated that biocatalysis in organic solvents refers to those systems in which the enzymes are suspended (or, sometimes, dissolved) in neat organic solvents in the presence of enough aqueous buffer (less than 5%) to ensure enzymatic activity. However, in the case of hydrolases water is also a substrate and it might be critical to find the water activity (a ) value to which the synthetic reaction (e.g. ester formation) can be optimized. Vahvety et al. [5] found that, in some cases, the activity of Candida rugosa lipase immobihzed on different supports showed the same activity profile versus o but a different absolute rate. With hpase from Burkholderia cepacia (lipase BC), previously known as lipase from Pseudomonas cepacia, and Candida antarctica lipase B (CALB) it was found that the enzyme activity profile versus o and even more the specific activity were dependent on the way the enzyme was freeze dried or immobihzed [6, 7]. A comparison of the transesterification activity of different forms of hpase BC or CALB can be observed in Tables 5.1 and 5.2, respectively. 

Biocatalysis is a key route to both natural and non-natural polysaccharide structures. Research in this area is particularly rich and generally involves at least one of the following three synthetic approaches 1) isolated enzyme, 2) whole-cell, and 3) some combination of chemical and enzymatic catalysts (i.e. chemoenzymatic methods) (87-90). Two elegant examples that used cell-fi-ee enzymatic catalysts were described by Makino and Kobayashi (25) and van der Vlist and Loos (27). Indeed, for many years, Kobayashi has pioneered the use of glycosidic hydrolases as catalysts for polymerizations to prepare polysaccharides (88,91). In their paper, Makino and Kobayashi (25) made new monomers and synthesized unnatural hybrid polysaccharides with regio- and stereochemical-control. Van der Vlist and Loos (27) made use of tandem reactions catalyzed by two different enzymes in order to prepare branched amylose. One enzyme catalyzed the synthesis of linear structures (amylose) where the second enzyme introduced branches. In this way, artificial starch can be prepared with controlled quantities of branched regions. 

An analysis of the commercialised biotransformations (Fig. 1) shows that in 50% of the processes only hydrolases are being used. This is in line with the analysis of Faber [4], who showed that about 60% of the research publications on biocatalysis deal with hydrolases. The reason for this is partly that making a molecule is more difficult than breaking a molecule. However, hydrolases are also used in the reverse mode, pulling the equilibrium towards synthesis by water removal during the reaction, for example... 

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