Organic solvents biocatalysts stability

TABLE 1 Effect of the Organic Solvent on Stability and/or Activity of Biocatalysts in Biphasic Media... 

Adsorption on solid matrices, which improves (at optimal protein/support ratios) enzyme dispersion, reduces diffusion limitations and favors substrate access to individual enzyme molecules. Immobilized lipases with excellent activity and stability were obtained by entrapping the enzymes in hydrophobic sol-gel materials [20]. Finally, in order to minimize substrate diffusion limitations and maximize enzyme dispersion, various approaches have been attempted to solubilize the biocatalysts in organic solvents. The most widespread method is the one based on the covalent linking of the amphiphilic polymer polyethylene glycol (PEG) to enzyme molecules [21]. 

Organic solvent Log P Biphasic medium Biocatalyst Reaction Biocatalyst stability Activity Reference... 

The volumetric ratio of the two liquid phases (j6 = Forg/ Faq) can affect the efficiency of substrate conversion in biphasic media. The biocatalyst stability and the reaction equilibrium shift are dependent on the volume ratio of the two phases [29]. In our previous work [37], we studied the importance of the nonpolar phase in a biphasic system (octane-buffer pH 9) by varying the volume of solvent. The ratio /I = 2/10 has been the most appropriate for an improvement of the yield of the two-enzyme (lipase-lipoxygenase) system. We found that a larger volume of organic phase decreases the total yield of conversion. Nevertheless, Antonini et al. [61] affirmed that changes in the ratios of phases in water-organic two-phase system have little effect upon biotransformation rate. 

The improvement of its activity and stability has been approach by the use of GE tools (see Refs. [398] and [399], respectively). A process drawback is the fact that the oxidation of hydrophobic compounds in an organic solvent becomes limited by substrate partition between the active site of the enzyme and the bulk solvent [398], To provide the biocatalyst soluble with a hydrophobic active site access, keeping its solubility in organic solvents, a double chemical modification on horse heart cytochrome c has been performed [400,401], First, to increase the active-site hydrophobicity, a methyl esterification on the heme propionates was performed. Then, polyethylene glycol (PEG) was used for a surface modification of the protein, yielding a protein-polymer conjugates that are soluble in organic solvents. 

A practical enzymatic procedure using alcalase as biocatalyst has been developed for the synthesis of hydrophilic peptides.Alcalase is an industrial alkaline protease from Bacillus licheniformis produced by Novozymes that has been used as a detergent and for silk degumming. The major enzyme component of alcalase is the serine protease subtilisin Carlsberg, which is one of the fully characterized bacterial proteases. Alcalase has better stability and activity in polar organic solvents, such as alcohols, acetonitrile, dimethylformamide, etc., than other proteases. In addition, alcalase has wide specificity and both l- and o-amino acids that are accepted as nucleophiles at the p-1 subsite. Therefore, alcalase is a suitable biocatalyst to catalyse peptide bond formation in organic solvents under kinetic control without any racemization of the amino acids (Scheme 5.1). 

Lactobacillus kefir (ADH E.C. 1.1.1.1) for use in organic solvents [11, 12]. Both biocatalysts are characterized by a very low stability in pure organic solvents or standard aqueous-organic two-phase systems [20], though their broad substrate ranges include many hydrophobic compounds [21, 22]. Figure 3.2.2 illustrates the denaturation of native BAL at the interface between a buffered aqueous solution and octanone. 

As previously observed for BAL, entrapment of ADH in PVA and its subsequent application in hexane as a standard organic solvent enabled the conversion of a number of interesting hydrophobic substrates (Table 3.2.3) by stabilizing the delicate biocatalyst against deactivating effects of the aqueous-organic interface. 

The toxic effect on biocatalytic activity and stability in two-phase reaction system media can be divided into two effects. The first one, called the molecular-toxicity effect, is a direct toxic effect of the solvent molecules, which are dissolved in the aqueous phase and interact with the biocatalyst, particularly with whole cells. The second one, which is created by the presence of an interface between the aqueous and the organic solvent phase, is called the phase-toxicity effect [2, 24]. 

Despite these advantages, native enzymes almost universally exhibit very low activities in organic solvents-often 4-5 orders of magnitude lower than in aqueous solutions. This loss in catalytic activity may be attributed to several factors, including a decrease in the polarity of the enzyme s microenvironment, the loss of critical water residues from the enzyme s surface, the decreased conformational mobility of the enzyme s structure, ground-state stabilization of hydrophobic substrates, and deactivation during the preparation of the biocatalyst for use in nonaqueous media,... 

One of the reactions catalyzed by esterases and lipases is the reversible hydrolysis of esters (Figure 19.1, Reaction 2). These enzymes also catalyze transesterilications and the desymmetrization of mew-substrates (vide infra). Many esterases and lipases are commercially available, making them easy to use for screening desired biotransformations without the need for culture collections and/or fermentation capabilities.160 In addition, they have enhanced stability in organic solvents, require no co-factors, and have a broad substrate specificity, which make them some of the most ideal industrial biocatalysts. Alteration of reaction conditions with additives has enabled enhancement and control of enantioselectivity and reactivity with a wide variety of substrate structures.159161164... 

There has been considerable effort directed toward the immobilization of both enzymes and whole cells in a wide array of formats.15 Initial attempts to immobilize enzymes on naturally derived supports such as charcoal were conducted early in the twentieth century and eventually led to the development of more robust biocatalysts immobilized on synthetic resins by the mid-1950s. Immobilization often confers a number of advantages relative to the free biocatalyst including ease of removal from the process stream, potential for reuse, improvements in stability, favorable alterations in kinetic parameters, suitability for continuous production and in some cases the ability to operate in organic solvents. The focus of this section is on the immobilization of enzymes, however, many of the same principles apply to whole cells, the primary difference being the fact that immobilized cells are often less stable than individual enzymes and may contain additional undesired enzyme activities. 

Adercruetz P and Mattiasson B. Aspects of Biocatalyst Stability in Organic Solvents. Biocatalysis 1987 1 99-108. 

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