Porous Pdms
Fig. 3. (A) SEM image of untreated SIRAN showing hollow pores in which the lipase-containing sol-gel material can be bound. (B) SEM image of a lipase SP 523 containing TMOS/PDMS (4:1) gel in the pores of SIRAN. (C) Approximate 100-fold magnification of the SEM image of (B) (19).
Lite Immobilisation Lipase
Fig. 4. Schematic view of noncovalent interactions between the gel matrix and the lipase (21).
Fig. 5. Activity of immobilized lipase PS (Amano) after repeated use in esterification reactions involving 1 + 2 ^ 3. (A) MTMS/PDMS (6:1) gel with entrapped lipase PS. (B) MTMS gel with entrapped lipase PS. (C) MTMS gel with adsorbed lipase PS (18,21).

Successful recycling of sol-gel lipase immobilizates derived from eight different lipases was demonstrated (18,21). For example, the entrapped lipase from Pseudomonas cepacia in MTMS or MTMS/PDMS gels were repeatedly used in batch esterification involving the model reaction of 1 and 2. After shaking the reaction mixture at 30°C for 23 h, the lipase-containing gels were recovered by filtration or centrifugation, washed with isooctane and pentane, and reused. In addition, the gels were washed with acetone after every fifth reaction. After a slight loss of enhanced activity, probably resulting from a loss of surface adsorbed lipase, enzyme activity remained constant for at least 30 reactions at about 85% of the original high value. Under the same conditions a control experiment in which the lipase was physically adsorbed to the hydrophobic gel showed that the material loses its activity completely after only a few reactions (see Fig. 5) (18,21). These experiments illustrate an important point, namely that immobilization by adsorption is certainly viable in this system (and in other types of immobilizates), but only for a very limited number of cycles involving reaction/recyclization. This phenomenon is not always reported in the literature.

Although the sol-gel lipase immobilizates were designed for application in nonaqueous media, they can also be used as heterogeneous catalysts for ester hydrolysis in aqueous medium (22). Another development concerns the possibility of magnetic separation as a means to recycle the lipase catalysts, specifically by incorporating magnetite (iron oxide) during the sol-gel process (23). Moreover, the concept of lipase entrapment in hydrophobic silicates has been extended to aerogels (24,25).

Significant progress was made possible by the development of second-generation sol-gel lipase immobilizates (26). They opened the way to heterogeneous cata lysts which are more active while at the same time retaining enhanced thermal and mechanical stability and the possibility to recycle efficiently. It involves the optimal choice of the alkyl group in the sol-gel precursor RSi(OCH3)3 (Fig. 2, 4) as well as the use of additives such as 18-crown-6, Tween-80, cyclodextrins, isopro-panol, and/or KCl. Such additives had been previously used to activate lipases in the absence of sol-gels (27-37). The most active lipase gels are based on w-butyl-or isobutylsilane precursors (Figs. 2, 4d,e) and contain either 18-crown-6 or Tween-80 as additives (26). Nine different lipases (PfL, BcL, MmL, AnL, CrL (type VII), CrL(L-3), TIL, PpL and PrL as described in Subheading 3.) were subjected to sol-gel immobilization of the second generation, and all of them displayed improved catalytic performance (26). In some cases, "double" immobilization (i.e., performing the sol-gel lipase entrapment not only in the presence of an appropriate additive, but also in the presence of a solid support such as SIRAN [see Fig. 3]) or Celite led to additional enhancement of activity as well as thermal and mechanical stability. Although this actually increases the amount of solid support (Celite plus attached sol-gel silicate) relative to the amount of lipase, less total heterogeneous catalyst needs to be employed weight-wise. This is because the enzyme activity in the model reaction is dramatically higher relative to the use of lipase powders. For example, in the case of the lipase TIL, the factor is 1391 (relative activity), corresponding to an activity of 1237 ^mol/ming gel and a specific activity of 8899 mmol/min g protein. Sol-gel encapsulation of lipases also enhances enantioselectivity in most reactions tested, as in the kinetic resolution of rac-6 (Table 1 and Fig. 6) (26).

It has been noted that in many protocols concerning biocatalysis (including lipases) in nonaqueous medium more enzyme than substrate is used weight-wise (38). When employing second-generation sol-gel lipase immobilizates as heterogeneous catalysts, this is not the case (26). In a typical preparative scale reaction, only about 250 mg of a lipase-containing silica gel are needed for 10 g of substrate, as demonstrated in the kinetic resolution of rac-9 (Fig. 7). This is all the more significant when recalling that most of the heterogeneous catalyst is in fact weight-wise the silicate matrix.

In summary, the second-generation sol-gel lipase immobilizates (26) are considerably more active and enantioselective than the original first-generation materials (17,18,21,39-43). They constitute industrially viable heterogeneous biocatalysts.

2. Materials

2.1. Lipase-Catalyzed Reactions

1. Lauric acid (Fluka, Switzerland).

3. rac-2-Octanol (Aldrich, Germany).

5. rac-2-Naphthyl-2-ethanol (Aldrich).

6. Isooctane (Fluka).

7. Toluene (Overlack, Germany).

Table 1

Kinetic Resolution of Alcohol3 Using Traditional Lipase-Powders and Sol-Gel Encapsulated Lipases as Catalysts, Vinyl Acetateb as the Acylating Agent, and Isooctane as Solvent (26)

Additives used Activity Specific activity Selectivity

Entry Lipase in sol-gel process (|J.mol/mm-g gel) (|J.mol/mm-g protein) Relative activity factor (E)

Table 1

Kinetic Resolution of Alcohol3 Using Traditional Lipase-Powders and Sol-Gel Encapsulated Lipases as Catalysts, Vinyl Acetateb as the Acylating Agent, and Isooctane as Solvent (26)

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