Biocatalysts examples

A considerable number of industrial bioconversions utilize covalently immobilized biocatalysts. Examples include Penicillin acy-lases V and G, aminoacylases, and aspartase. In some cases the biocatalyst is immobilized through cross-linking and in others the catalyst is captured by a reactive resin. Covalent immobilization often leads to extremely stable catalysts with high potential for reuse and... 

The Michaelis-Menten equation (8.8) and the irreversible Uni Uni kinetic scheme (Scheme 8.1) are only really applicable to an irreversible biocatalytic process involving a single substrate interacting with a biocatalyst that comprises a single catalytic site. Hence with reference to the biocatalyst examples given in Section 8.1, Equation (8.8), the Uni Uni kinetic scheme is only really directly applicable to the steady state kinetic analysis of TIM biocatalysis (Figure 8.1, Table 8.1). Furthermore, even this statement is only valid with the proviso that all biocatalytic initial rate values are determined in the absence of product. Similarly, the Uni Uni kinetic schemes for competitive, uncompetitive and non-competitive inhibition are only really applicable directly for the steady state kinetic analysis for the inhibition of TIM (Table 8.1). Therefore, why are Equation (8.8) and the irreversible Uni Uni kinetic scheme apparently used so widely for the steady state analysis of many different biocatalytic processes A main reason for this is that Equation (8.8) is simple to use and measured k t and Km parameters can be easily interpreted. There is only a necessity to adapt catalysis conditions such that... 

This is essentially identical with the form of the Michaelis-Menten equation (8.8), although the meaning of the corresponding biocatalytic parameters is slightly modified. The kinetic scheme upon which this derivation is based is clearly limited to a single-substrate biocatalyst that operates with two independent catalytic sites. Such a biocatalyst could be monomeric with two catalytic sites, or else homodimeric with one catalytic site per subunit. With reference to the biocatalyst examples described in Section 8.1, the chemical biology reader should be able to see that Equation (8.25) and the Uni Uni kinetic scheme for two catalytic sites seems... 

The King-Altman approach described here can be summarised as a process in which an original rate equation (such as Equation (8.53)) is customarily developed, converted into a coefficient form (such as Equation (8.54)) and from there simplified to steady state kinetic forms (for example Equations (8.62) and (8.63)) by algebraic manipulation and Haldane simplification. This King-Altman approach is an approach that can be generalised for the derivation of most steady state kinetic equations based upon most complex kinetic schemes. Clearly these derivations can be substantial, but we shall not bother to reproduce these here except to cover a few important examples of particular relevance to the biocatalyst examples described in Section 8.1. 

Biosensors are traditionally divided into two main classes. One class includes biosensors that use a biological receptor, and the other uses a biocatalyst. Examples of bio-receptors include antibodies, binding proteins, and lectins. A critical evaluation of bio-receptor-based biosensors has recently been published (1). 

The use of biocatalysts for the selective introduction and cleavage of esters is vast and has been extensively reviewed." Therefore only a few examples of the types of transformations that are encountered in this area of protective group chemistry will be illustrated to show some of the basic transformations that have appeared in the literature. The selective... 

When ionic liquids are used as replacements for organic solvents in processes with nonvolatile products, downstream processing may become complicated. This may apply to many biotransformations in which the better selectivity of the biocatalyst is used to transform more complex molecules. In such cases, product isolation can be achieved by, for example, extraction with supercritical CO2 [50]. Recently, membrane processes such as pervaporation and nanofiltration have been used. The use of pervaporation for less volatile compounds such as phenylethanol has been reported by Crespo and co-workers [51]. We have developed a separation process based on nanofiltration [52, 53] which is especially well suited for isolation of nonvolatile compounds such as carbohydrates or charged compounds. It may also be used for easy recovery and/or purification of ionic liquids. 

When compared to traditional chemical synthesis, processes based on biocatalysts are generally less reliable. This is due, in part, to the fact that biological systems are inherently complex. In bioprocesses involving whole cells, it is essential to use the same strain from the same culture collection to minimise problems of reproducibility. If cell free enzymes are used the reliability can depend on the purity of the enzyme preparation, for example iso-enzyme composition or the presence of other proteins. It is, therefore, important to consider the commercial source of the enzyme and the precise specifications of the biocatalyst employed. 

The specificity of biocatalysts also extends to site specificity (regiospecificity). This means that if several functional groups of one type are present on the molecule, only one specific position will be affected. An example of this is the microbial oxidation of D-soibitol to L-soibose, a key step in the synthesis of vitamin C (Figure 2.4). 

Photochemical methods [6] have been developed to provide an environmentally friendly system that employs light energy to regenerate NAD(P)H, for example, by the use of a cyanobacterium, a photo synthetic biocatalyst. Using the biocatalysts, the... 

A dried cell mass is often used as a biocatalyst for a reduction since it can be stored for a long time and used whenever needed, without cultivation. One convenient method of drying the cell mass is acetone dehydration. For example, dried cells of G. 

Dynamic kinetic resolution of a-alkyl-P-keto ester was conducted successfully using biocatalysts. For example, baker s yeast gave selectively syn(2R, 3S)-product [29a] and the selectivity was enhanced by using selective inhibitor [29b] or heat treatment of the yeast [29c]. Organic solvent was used for stereochemical control of G. candidum [29d]. Plant cell cultures were used for reduction of 2-methyl-3-oxobu-tanoate and afforded antialcohol with Marchantia [29e,f] and syn-isomer with Glycine max [29f]. 

Other biocatalysts were also used to perform the dynamic kinetic resolution through reduction. For example, Thermoanaerobium brockii reduced the aldehyde with a moderate enantioselectivity [30b,c], and Candida humicola was found, as a result of screening from 107 microorganisms, to give the (Jl)-alcohol with 98.2% ee when ester group was methyl [30dj. 

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. 

Recently, recombinant biocatalysts obtained using Escherichia coli cells were designed for this process. The overexpression of all enzymes required for the process, namely, hydantoinase, carbamoylase, and hydantoin racemase from Arthrobacter sp. DSM 9771 was achieved. These cells were used for production of a-amino acids at the concentration of above 50 g 1 dry cell weight [37]. This is an excellent example presenting the power of biocatalysis with respect to classical catalysis, since a simultaneous use of three different biocatalysts originated from one microorganism can be easily achieved. 

Lipases are the enzymes for which a number of examples of a promiscuous activity have been reported. Thus, in addition to their original activity comprising hydrolysis of lipids and, generally, catalysis of the hydrolysis or formation of carboxylic esters [107], lipases have been found to catalyze not only the carbon-nitrogen bond hydrolysis/formation (in this case, acting as proteases) but also the carbon-carbon bond-forming reactions. The first example of a lipase-catalyzed Michael addition to 2-(trifluoromethyl)propenoic acid was described as early as in 1986 [108]. Michael addition of secondary amines to acrylonitrile is up to 100-fold faster in the presence of various preparations of the hpase from Candida antariica (CAL-B) than in the absence of a biocatalyst (Scheme 5.20) [109]. 

This example involves the same diffusion-reaction situation as in the previous example, ENZSPLIT, except that here a dynamic solution is obtained, using the method of finite differencing. The substrate concentration profile in the porous biocatalyst is shown in Fig. 5.252. 

Several micro-organism- or enzyme-catalyzed reactions are performed in two-phase systems (Table 1 and 4). The examples given illustrate the advantages of the procedure which may be of practical interest in technological applications of biocatalysts. 

In addition to the enzymatic hydrolysis of esters, there also ample examples where an epoxide has been cleaved using a biocatalyst. As described by the Faber group [19], reaction of the ( )-2,3-disubstituted ds-chloroalkyl epoxide roc-8-40 with a bacterial epoxide hydrolase (BEH), led to the formation of vie-diol (2 ,3S)-8-41 (Scheme 8.11). The latter underwent a spontaneous cydization to give the desired product (2i ,3i )-8-42 in 92 % ee and 76 % yield. The same strategy was used with the homologous molecule rac-8-43, which afforded the THF derivative (2R,3R)-S-4S in 86% ee and 79% yield. 

The reduction of several ketones, which were transformed by the wild-type lyophilized cells of Rhodococcus ruber DSM 44541 with moderate stereoselectivity, was reinvestigated employing lyophilized cells of Escherichia coli containing the overexpressed alcohol dehydrogenase (ADH- A ) from Rhodococcus ruber DSM 44541. The recombinant whole-cell biocatalyst significantly increased the activity and enantioselectivity [41]. For example, the enantiomeric excess of (R)-2-chloro-l-phenylethanol increased from 43 to >99%. This study clearly demonstrated the advantages of the recombinant whole cell biocatalysts over the wild-type whole cells. 

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