Enzymes, immobilized lipase-Celite

Enzyme activity for the polymerization of lactones was improved by the immobilization on Celite [93]. Immobilized lipase PF adsorbed on a Celite showed much higher catalytic activity than that before the immobilization. The catalytic activity was further enhanced by the addition of a sugar or poly(ethylene glycol) in the immobilization. Surfactant-coated lipase efficiently polymerized the ring-opening polymerization of lactones in organic solvents [94]. 

The rather complex furylvinylcarbinol derivative 76 shown in Scheme 4.28 was required in enantiopure form as a key intermediate in the synthesis of the natural product cneorin. The carbinol moiety is heavily substituted with sterically demanding groups. Therefore attempts to resolve the furylvinylcarbinol with CALB or lipase PS-II led to very slow reactions. However, the rarely used enzyme Candida antarctica lipase A (CALA), which is known to act on sterically hindered substrates offers an alternative. Thus acylation of the furylvinylcarbinol 76 with 2,2,2-trifluoroethyl butanoate catalyzed by CALA (immobilized on celite with sucrose at pH 7.9) furnished the enantiomerically enriched propanoate of S-76 and R-76 (Scheme 4.28) [90]. Small-scale experiments gave E > 300. 

Immobilized lipase showing high catalytic activity toward the macrolide polymerization was demonstrated.169 The immobilization of lipase PF on Celite greatly improved the rate of the DDL polymerization. Catalytic activity was further enhanced by the addition of a sugar or polyethylene glycol) (PEG) during immobilization. A surfactant-coated enzyme was used... 

Apart from CALB other enzymes were shown to be able to successfully polymerize lactones. For instance Lipase PS-30, immobilized on Celite, was used as catalyst to study PDL-ROP under bulk reaction conditions. Poly(PDL) with M = 62000 and PDI 1.9 was reported [76]. Gross and coworkers could show that Humicola insolens cutinase (HiC) showed a high catalytic activity for enzymatic ROP of e-CL and PDL [87]. Poly(e-CL) with M n = 16000 (M /lVf = 3.1), in >99% yields was produced in bulk (70 °C, 24h) with 0.1% w/w immobilized HiC. Furthermore, using immobilized HiC in toluene (70 °C, 24h), PDL was converted to poly(PDL) (99% yield) with M = 44600 and Mw/M = 1.7. 

It is known in the literature that some enzymes can catalyze the acylation reaction of polysaccharides (9,10). Not surprisingly, lipases can serve this function (9) because lipases are known to mediate ester hydrolysis or synthesis. Yet, some proteases can also catalyze this reaction (10). For example, Alcalase protease immobilized on Celite can transfer the acrylic group onto hydroxy-ethylcellulose (HEC) to form an ester(9a) (Figure 2). Thus, in this case Alcalase appears to have broad substrate specificity and can work well with esters. 

The catalyst was the unsupported lipase from Pseudomonas cepacia. The reaction was as selective in SCCO2 as it was in hexane but was much slower in SCCO2. The S ester formed preferentially, in 46% e.e. (after 63% conversion), leaving the R acid in 78% e.e. Later experiments compared the eflfectiveness of the crude enzyme with the epoxy- and Celite-stabilized enzymes (82). All three were stable to exposure to SCCO2 and greater rates of reaction were found with the immobilized versions, but the conversions dropped greatly when the catalyst was reused. 3-Hydroxy esters can be used as intermediates for the preparation of optically active pharmaceuticals such as propranolol, a (5-blocker (82). 

The most important factor for adsorption is the choice of immobilization matrix. Several commercially available matrices commonly employed for nonaqneous enzymology are listed in Table 8.3. They consist of macroporons millimeter-sized particulates that are hydrophilic with the exception of Accnrel (polypropylene). Celite (diatomaceons earth) is probably, the most commonly employed matrix of those listed in Table 8.3. In addition, a few enzymes are commercially available in immobilized forms, inclnding Lipozyme IM and Novozyme from Novo-Nordisk, the Chirazyme prodnct line from Roche Molecular Biochemicals, and Pseudomonas cepacia lipase immobilized in Sol-Gel AK (Fluka). 

FIGURE 8.2. Transesterification rate of Pseudomonas sp. lipase immobilized by adsorption onto Celite as a function of enzyme loading. (Redrawn from Bovara, R. et al., Biotechnol. Lett., 15, 169-174, 1993. With permission. 

A lipase has been used to convert solid triolein (glycerol trioleate) to the monooleate by treatment with glycerol at 8°C.75 Other lipases have been used in the hydrolysis and transesterification of oils, as well as in the esterification of fatty acids without solvents.76 Peptides can be produced from eutectic mixtures of amino acid derivatives with the addition of a small amount of solvent.77 Immobilized sub-tilisin and thermolysin were used with 19-24% water or an alcohol to produce polypeptides. Subtilisin on celite (a di-atomaceous earth) was used to convert a mixture of L-phenylalanine ethyl ester and L-leucinamide containing 10% triethyleneglycol dimethyl ether to L-phenylala-nineleucinamide in 83% yield. Addition of 30% 2 1 ethanol/water reduced the time needed from 40 to 4 h. The enzyme could be used three more times. These reaction mixtures contained 0.13-0.75 g peptide per g reaction mixture compared with 0.015-0.035 when the reaction was... 

Covalent immobilization of CRL onto an epoxy-activated macroscopic carrier leads to selective monoalkylation of the lysine amino residues which are involved in the deactivation reaction with retention of the positive charge. In contrast to the native enzyme, the immobilized enzyme is inert towards the formation of Schiff bases, which results not only in a greatly stabilized activity but also in a significant enhancement in selectivity [158]. The addition of a molecular sieve to the medium in order to trap acetaldehyde seems to have some benefit [159,160]. Alternatively, the lipase may be stabilized by adsorption onto Celite [161]. 

Various lipases can be used, the most employed being Candida antartica lipase A (CAL-A). Nevertheless, the best results have been obtained with CAL-B immobilized on a Celite R-633 support, yielding 97% of conversion in the synthesis of mandelonitrile acetate 12 with an ee of 98%. Many applications can be envisaged considering that numerous hydrophobic compounds, from aliphatic chains to various aromatics, can be used as substrates, and both (R)- and (5)-enantiomers can be obtained by an appropriate choice of the enzyme (examples of (R)-enantiomers with CAL-A,Burkholderia cepiaca lipase [BCL], Candida rugosa lipase [CRL], " S) with CAL-B, BCL, and porcine pancreas lipase [PPL] " ). 

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