Enzymes Upases

In Section 8.3.3 several examples of cascade systems involving homogeneous catalytic and enzymatic reactions were presented. There are also examples when heterogeneous catalytic reactions are combined with enzymatic catalysis. Fig. 8.35 shows a combination of a heterogeneous catalytic reaction (hydrogenation of a ketone over a supported metal catalyst) and an enzymatic one (acylation of obtained R-alcohol into the corresponding to R-acetate over an immobilized enzyme, Upase). 

Hydrolytic enzymes such as esterases and Upases have proven particularly useful for asymmetric synthesis because of their abiUties to discriminate between enantiotopic ester and hydroxyl groups. A large number of esterases and Upases are commercially available in large quantities many are inexpensive and accept a broad range of substrates. 

For the kinetic resolution of racemic a-acetoxyamide 6 several native enzymes were used (Scheme 5.5). The native Upases from Pseudomonas cepacia (PCL) and porcine pancreas (PPL) showed the highesL although stiU unsatisfactory, enantios-electivity ( = 5.1 and 3.5, respectively). Upon immobilization into a solgel matrix, the enantioselectivity of PCL was improved significantly to 30.5. The covalent immobilization on Eupergit increased the enantioselectivity even more (34.0) [23]. 

Certain enzymes, proenzymes, and their substrates are present at all times in the circulation of normal individuals and perform a physiologic function in the blood. Examples of these functional plasma enzymes include lipoprotein Upase, pseudocholinesterase, and the proenzymes of blood coagulation and blood clot dissolution (Chapters 9 and 51). The majority of these enzymes are synthesized in and secreted by the liver. 

The first P-chiral hydroxy phosphoryl compounds that were enzymatically resolved into enantiomers were o-hydroxyaryl phosphines and their oxides 75. The resolution was achieved via enzyme-assisted hydrolysis of their O-acetyl derivatives 74, the most effective enzymes being CE and Upase from C. rugosa (CRL) (Equation 35). The highest enanfioselectivity was observed in the case of naphthyl derivatives (Equation 36), having a P=0 moiety. ... 

The glycerol produced by the action of hormone-sensitive Upase in the adipose tissue cannot be utilized by adipose tissue itself. Adipose ceUs lack the enzyme glycerol kinase, which is necessary to convert glycerol to glycerol phosphate. 

Triacylglycerol Upases [EC 3.1.1.3] (also known as triglyceride lipases, tributyrases, or simply as lipases) catalyze the hydrolysis of a triacylglycerol to produce a diac-ylglycerol and a fatty acid anion. The pancreatic enzyme acts only on an ester-water interface the outer ester links in the substrate are the ones which are preferentially... 

The stability of the ester surfactants against enzymatic hydrolysis by two different microbial Upases, Mucor miehei lipase (MML) and Candida antarc-tica lipase B (CALB) added separately to the surfactant solutions, was also investigated, see Fig. 5 [19]. It is obvious that hydrolysis of the unsubstituted surfactant is much faster with both CALB and MML than that of the substituted surfactants, i.e., increased steric hindrance near the ester bond leads to decreased hydrolysis rate. Since the specificity of the enzyme against its substrate is determined by the structure of the active site, it can be concluded, as expected, that the straight chain surfactant most easily fits into the active site of both enzymes. 

It is not known which molecular features of Upases keep them active at low water activity. It is worth pointing out that when they are used to catalyze various reactions, such as hydrolysis, reversed hydrolysis, and transesterification reactions, the water activity dependence is similar in the different reactions [26, 28]. The same is true for phospholipase A2 [29]. This shows that the effect of water on the enzyme is more important than the effect of water as a reactant when determining the reaction rate at different water activities. 

Finally, the purity-related performance of the enzyme preparation would appear to be a constant throughout a series of measurements. This, however, may not be the case when several enzyme species are present and different activities are expressed as a function of the medium composition [100]. Although it would appear that this source of error is abolished by the present-day availability of highly pure enzyme preparations (but see also [101, 102]), the intrinsic properties of i.e. Upases may lead to different E-values as a result of interfacial activation [103] and the conformation of the lid structure of lipases [56]. 

Naturally occurring Upases are (R)-selective for alcohols according to Kazlauskas rule [58, 59]. Thus, DKR of alcohols employing lipases can only be used to transform the racemic alcohol into the (R)-acetate. Serine proteases, a sub-class of hydrolases, are known to catalyze transesterifications similar to those catalyzed by lipases, but, interestingly, often with reversed enantioselectivity. Proteases are less thermostable enzymes, and for this reason only metal complexes that racemize secondary alcohols at ambient temperature can be employed for efficient (S)-selective DKR of sec-alcohols. Ruthenium complexes 2 and 3 have been combined with subtilisin Carlsberg, affording a method for the synthesis of... 

The seminal work of Klibanov in the early 1980s [46,47] made it clear that enzymes can be used in hydrophobic organic solvents, although at the price of a severely reduced reaction rate [48, 49]. Indeed, many Upases, as well as some proteases and acylases, are so stable that they maintain their activity even in anhydrous organic solvents. This forms the basis for their successful application in non-hydrolytic reactions, such as the (enantioselective) acylation of alcohols and amines, which now are major industrial applications [50]. 

Three proteases account for almost all sales to the dairy industry, ie, chymosin extracted from calves stomachs, chymosin produced by fermentation, and substitutes also produced by fermentation. Four different types of enzyme are used in the detergent industry, ie, proteases, amylases, cellulases, and Upases. Cellulases and Upases have only recently been introduced. Table 8 shows a breakdown of estimated woddwide sales consumption by product types. 

R, Braste, T. Friedrich, R. Kurth.E Mcnkd-Ccmen. H. Retlmnaier, and S. UbkowskL Use of Upase derived from t seudomonas sp, PSm for treatment of enzyme-deficiency digestive diBoiders, Patent WO 9300924. 

The alkylation chemistry described above can also be appUed with fluorescein to yield the corresponding fluorescein mono-ethers such as 30 as green fluorescent probes for Upases [41]. These substrates are water soluble due to the anionic car-boxylate on the fluorescein group and can be used in pure aqueous buffer without any co-solvent This latter class of substrates reacts extremely fast and specificaUy with the enzymes, with assay times under 1 min. An activity fingerprinting study... 

The assay is carried out at a low substrate concentration due to the limited solubility and very low Ku values for this substrate. Most interestingly, the substrate is extremely resistant towards non-specific hydrolysis, in particular at alkaline pH. This allows one to screen enzymes under strongly basic conditions, as illustrated for the Roche Upase L8 (Scheme 1.10). A survey of various Upases and esterases at pH 11 showed that most enzymes in fact lose their activity under these conditions. 

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