Organic solvents enzyme stabilization

Takahashi H, Li B, Sasaki T et al (2000) Catalytic activity in organic solvents and stability of immobilized enzymes depend on the pore size and surface characteristics of mesoporous silica. Chem Mater 12 3301-3305... 

For enzymes another point can be important Not all pure enzymes are stable in solution. In particular in organic solvents enzymes can be very unstable (4). The stability of enzymes can be improved by genetic modifications, by chemical modification, and most easily and without specialist equipment by immobilization. Furthermore, immobibzation opens up the possibility of improving other properties of the enzyme, such as its activity, substrate specificity, and enantioselectivity. Although the underlying principles are not always understood enzyme immobilization is a powerful tool for the improvement of activity and enzyme stability, specificity, and selectivity. 

To sum up, enzymes in ionic liquids could maintain their activity over a much longer period than in molecular organic solvents. This stabilization has been explained on the basis of the interaction of the ionic liquid ions as well as higher viscosity of ionic liquids with respect to conventional organic solvent, which could cause slower migration of protein domains from the active conformation into the inactive one. [33]. 

The discussion in the above Sect. 10.3 gives a clear indication that with a few exceptions in most ILs, enzymes remain active and perform better or comparable to conventional organic solvents. Enzyme activity is closely related to the structure and conformational change at the microenviromnent of the enzyme active site. As discussed before (Sect. 10.2) microenviromnents of the enzyme active site is affected by ILs, which has complicated solvation characteristics due to different interactions of large organic cations and anionic counterparts of ILs with the enzyme. So it is important to understand the stability of enzymes in ILs. 

The enzyme concentrate is dried or dispersed in an organic solvent and stabilized by preservatives for use in liquid detergents. 

R Femandez-Lafuente, CM Rosell, JM Guisan. Enzyme reaction engineering synthesis of antibiotics catalysed by stabilized penicillin G acylase in the presence of organic solvents. Enzyme Microb Technol 13 898-905, 1991. 

As with organic solvents, proteins are not soluble in most of the ionic liquids when they are used as pure solvent. As a result, the enzyme is either applied in immobilized form, coupled to a support, or as a suspension in its native form. For production processes, the majority of enzymes are used as immobilized catalysts in order to facilitate handling and to improve their operational stability [24—26]. As support, either inorganic materials such as porous glass or different organic polymers are used [27]. These heterogeneous catalyst particles are subject to internal and external... 

In the first publication describing the preparative use of an enzymatic reaction in ionic liquids, Erbeldinger et al. reported the use of the protease thermolysin for the synthesis of the dipeptide Z-aspartame (Entry 6) [34]. The reaction rates were comparable to those found in conventional organic solvents such as ethyl acetate. Additionally, the enzyme stability was increased in the ionic liquid. The ionic liquid was recycled several times after the removal of non-converted substrates by extraction with water and product precipitation. Recycling of the enzyme has not been reported. It should be noted, however, that according to the log P concept described in the previous section, ethyl acetate - with a value of 0.68 - may interfere with the pro-... 

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]. 

Enzyme Kinetics and Stability Kinetic studies, carried out mostly with hydrolases, have shovm that enzymes in organic solvents follow conventional models [12a, 22]. 

Several reports have indicated that enzymes are more thermostable in organic solvents than in water. The high thermal stability of enzymes in organic solvents, especially in hydrophobic ones and at low water content, was attributed to increased conformational rigidity and to the absence of nearly all the covalent reactions causing irreversible thermoinactivation in water [23]. 

Thermostability of enzymes increases in apolar organic solvents increasing the stability and rigidity of the molecules. This effect is probably due to low-water activity [130]. 

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. 

Suspending enzyme in polymer solution instead of in pure organic solvent not only simplifies preparation of the casting solution, the enzyme suspensions became more uniform and stable. It was also found that at certain concentrations (enzyme, polyelectrolyte, and water) the resulting membranes exhibited extremes in both stability and... 

Unlike many other enzymes, the subtilisins are fairly stable towards e.g. organic solvents, anionic surfactants, high temperatures and high pH. This makes the subtilisins very suitable as detergent proteases. But despite this fact, stabilization of these protease enzymes in liquid detergents remains a major issue. 

Efficient biocatalysis in neat organic solvent depends on the careful choice of the method of dehydrated enzyme preparation and solvent used. Optimization of these factors towards a given transformation is often known as catalyst formulation and solvent, or medium, engineering respectively, both of which will be briefly discussed below. Catalyst engineering which also provides a powerful method of improving activity and stability, is discussed in Chapter 2. 

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). 

The hydrolysis of peptide bonds catalyzed by the serine proteases has been the reaction most extensively studied by low-temperature trapping experiments. The reasons for this preference are the ease of availability of substrates and purified enzymes, the stability of the proteins to extremes of pH, temperature, and organic solvent, and the existence of a well-characterized covalent acyl-enzyme intermediate. Both amides and esters are substrates for the serine proteases, and a number of chromo-phoric substrates have been synthesized to simplify assay by spectrophotometric techniques. 

Observations carried out on lysozyme in mixed solvents as a function of temperature demonstrate that lysozyme-catalyzed lysis can he performed under such abnormal conditions and that the reaction can be quenched at subzero temperatures and resumed by heating. The problem is now to check whether one can obtain an enzyme-substrate complex (in this case lysozyme-oligosaccharide) stabilized at low temperature. Such a complex can be detected by differential absorption spectroscopy. Difference spectra in the UV region (240-320 nm) were recorded. The reference cell contained a solution of lysozyme (1.39 x 10 M) and the sample cell contained a solution of lysozyme at the same concentration plus substrate, here hexa-NAG (1.66 x lO " M). The buffer used is acetate at pH 5 plus organic solvent. Also, difference spectra have been determined in the presence of the small-chain sac-... 

The presence of a covalent acyl-enzyme intermediate in the catalytic reaction of the serine proteases made this class of enzymes an attractive candidate for the initial attempt at using subzero temperatures to study an enzymatic mechanism. Elastase was chosen because it is easy to crystallize, diffracts to high resolution, has an active site which is accessible to small molecules diffusing through the crystal lattice, and is stable in high concentrations of cryoprotective solvents. The strategy used in the elastase experiment was to first determine in solution the exact conditions of temperature, organic solvent, and proton activity needed to stabilize an acyl-enzyme intermediate for sufficient time for X-ray data collection, and then to prepare the complex in the preformed, cooled crystal. Solution studies were carried out in the laboratory of Professor A. L. Fink, and were summarized in Section II,A,3. Briefly, it was shown that the chromophoric substrate -carbobenzoxy-L-alanyl-/>-nitrophenyl ester would react with elastase in both solution and in crystals in 70 30 methanol-water at pH 5.2 to form a productive covalent complex. These... 

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