Practical Protocols for Lipase Immobilization Via Sol Gel Techniques

Manfred T. Reetz


Lipases can be efficiently entrapped in the pores of hydrophobic silicates by a simple and cheap sol-gel process in which a mixture of an alkylsilane [RSi(OCH3)3 and Si(OCH3)4] is hydrolyzed under basic conditions in the presence of the enzyme. Additives such as iso-propanol, polyvinyl alcohol, cyclodextrins, or surfactants enhance the efficiency of this type of lipase immobilization. The main area of application of these heterogeneous biocatalysts concerns esterification or transesterification in organic solvents, ionic liquids, or supercritical carbon dioxide. Rate enhancements (relative to the traditional use of lipase powders) of several orders of magnitude have been observed, in addition to higher thermal stability. The lipase immobilizates are particularly useful in the kinetic resolution of chiral esters, enantioselectivity often being higher than what is observed when using the commercial forms of these lipases (powder or classical immobilizates). Thus, because of the low price of sol-gel entrapment, the excellent performance of the lipase immobilizates, and the ready recyclability, this method is industrially viable.

Key Words: Lipases; sol-gel immobilization; esterification; transesterification; thermal stability; kinetic resolution; enantioselectivity; recyclability.

1. Introduction

A wide variety of enzymes are available to the practicing organic chemist for many different transformations (1,2). A milestone in the application of enzymes as catalysts for synthetic organic chemistry was the discovery that numerous enzymes can be used in nonaqueous media, allowing transformations of interest to organic chemists to be performed that were not possible in the natural aqueous environment (3). An example of significant synthetic importance is the use of lipases (EC as catalysts in organic solvents. These enzymes are the most used biocatalysts in synthetic organic chemistry, catalyzing the hydrolysis of carboxy-

lic acid esters in aqueous medium, or the reverse reaction (esterification), as well as transesterification in organic solvents (1-4). When working in organic media, nucleophiles other than alcohols can be used (e.g., amines or H2O2 affording amides and acid peroxides). Numerous examples involving enantioselectivity in the production of chiral alcohols, amines, and carboxylic acids have been reported. Lipases are structurally characterized by a so-called lid. When hydrophobic substrates interact with certain hydrophobic regions of a lipase, the lid opens and thus exposes the active site (serine) in a process called interfacial activation (4-8).

In the case of reactions in organic solvents (which are most often used), commercially available lipase powders are often employed. In spite of the obvious advantages in such simple protocols, several drawbacks need to be considered, primarily the considerably reduced lipase activities relative to those observed in aqueous medium and the extreme difficulty in recycling the enzyme. Thus, for real (industrial) applications of lipases some form of immobilization that not only allows for efficient separation and re-use of the enzyme but also leads to a significant enhancement of catalyst activity is necessary. Several approaches have been described in this book (see Chapters 2 and 13). The present chapter focuses on the entrapment of lipases in hydrophobic sol-gel silicates.

Sol-gel encapsulation has proven to be a particularly easy and effective way to immobilize enzymes (9). Following isolated reports describing specific examples (10,11), it was the seminal work of Avnir and coworkers that led to the generalization of this technique (9,12-14). Sol-gel techniques involve the acid- or base-catalyzed hydrolysis of tetraalkoxysilanes [Si(OR)4] (15,16). Mechanistically, the silane-precursor undergoes hydrolysis and cross-linking condensation with formation of a SiO2 matrix in which the enzyme is encapsulated. This type of encapsulation works well for a number of enzymes (9,12-14). However, in the case of lipases, materials were obtained that showed disappointingly low enzyme activities, as measured by the rate of the model reaction involving the esterification of lauric acid (Fig. 1, 1), by n-octanol (2) in isooctane as solvent (17). Only a 5 to 10% activity relative to the traditional use of the respective lipase powder was observed, equivalent to relative rates of 0.05 to 0.1.

It was speculated that the microenvironment in SiO2 may be too polar, and therefore mixtures of Si(OCH3)4 (Fig. 2, 5) and alkylsilanes of the type RSi(OCH3)3 (4) or polydimethylsiloxane (PDMS) having nonhydrolyzable lipophilic alkyl groups (R) were tested (17). This strategy was developed because the silicon oxide matrix is now hydrophobic, which can facilitate or simulate a type of interfacial activation of the entrapped lipase. Basic catalysts such as NaF were used for the sol-gel process, because such conditions lead to large pores in the silicate matrix. Indeed, dramatically improved relative lipase activities typically amounting to 200 to 800% were observed in the model reaction, which corresponds to an enhancement of relative enzyme activity by a factor ranging from two to eight with respect to the traditional use of the corresponding lipase powder (lyophilizate) (17,18). Relative activity is defined as [^(immobilized lipase)/ v(commercial lipase)], where v is the initial rate of the reaction in each case. A pronounced increase in thermal stability was also observed. In most cases the optimal ratio of RSi(OCH3)3 to Si(OCH3)4 turned out to be about 5:1, although it was not possible to present an experimental protocol that was completely general.

Sol Gel Lipases

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