Lyophilized enzymes

Most enzyme powders are prepared by lyophilisation (freeze drying). However, the lyophilization procedure might inactivate the enzyme to some extent. To avoid this and thereby increase the activity of lyophilized enzymes in dry organic solvents, the lyophilization can be carried out in the presence of lyoprotectants such as sorbitol (Dabulis and Klibanov, 1993). The inactivation is believed to be caused at least partly by a reversible conformational change in the enzyme. This process can be reversed and the enzyme reactivated by the addition of small amoimts of water (Dabulis and Klibanov, 1993). 

Immobilization of enzymes to solid supports can increase activity over a wide range of solvents [78]. As seen in Table 3.2, the transesterification of N-acetyl-L-phenylalanine ethyl ester (APEE) with 1-propanol by a-chymotrypsin (Scheme 3.2) immobilized to glass is 1-2 orders of magnitude higher than that of the free, lyophilized enzyme. 

Table 3.2 Values of (kc /Km)app (M 1 min-1) x 100 for freely suspended and glass-immobilized a-chymotrypsin in various solvents. In all solvent conditions, the enzyme is 1-2 orders of magnitude more active than the free, lyophilized enzyme [78].

Lyophilized enzymes have a pH memory, meaning that the activity of the enzyme in organic solvent parallels its pH-activity profile of the aqueous solution from which it was lyophilized [36, 79-81]. However, very often acidic or basic mixtures within a nonaqueous reaction mixture such as reactant, products, or impurities, can disrupt this delicate protonation state, leading to changes in catalytic activity. To counteract this potential problem, solid-state buffers have been developed to protect the enzyme s protonation state in the nonaqueous environment [53, 82]. These solid-state buffers contain pairs of crystalline solids that can be intercon-... 

Khmelnitsky et al. were the first to observe the activating effects salt showed on enzymes in the nonaqueous environment [88]. As shown in Figure 3.7, the transesterification activity of the serine protease subtilisin Carlsberg in anhydrous solvents is strongly dependent on the KC1 content in a lyophilized enzyme preparation and increases sharply as the salt content is increased. This increase in activity was determined to be a result primarily of an increase in kcat and not a decrease in Km, as shown in (Table 3.4). 

Figu re 3.9 The catalytic efficiency, kca,/Km (IVT s-1), of subtilisin Carlsberg activated by various sodium (O) and potassium ( ) salts. Values represent the average of four trials from lyophilized enzyme samples prepared independently, and the... 

Furthermore, the type of enzyme formulation (free enzyme, immobilized enzyme, or whole cells) plays a key role in determining the progress of the overall reaction. For most applications, lyophilized enzyme powders have been used with good results presumably they dissolve into the liquid phase. When poorly soluble products are formed, the enzyme can be recovered by washing with water [52]. For co-factor-dependent reactions permeabilized cells may be used [44]. When using immobilized enzymes, it has been demonstrated that the chemical nature and the pore size of the support are very important parameters to consider [8, 41]. 

Catalytic activities of a-chymotrypsin and Subtilisin Carlsberg in various hydrous organic solvents were measured as a function of how the enzyme suspension had been prepared (Ke, 1998). Direct suspension of the lyophilized enzyme in the solvent containing 1% water was compared with precipitation of the same enzyme from its aqueous solution by a 100-fold dilution with anhydrous solvent. The reaction rate in a given non-aqueous enzymatic system was found to depend on the nature of both enzyme and solvent, but to depend strongly on the mode of enzyme preparation. 

The common mode of suspending lyophilized enzymes in organic solvents containing very little water results in just a low specific activity. Much higher specific activity can be achieved if the enzymes are dissolved in water first and then diluted with anhydrous organic solvent to the same water content (Dai, 1999) the lower the water content of the medium, the greater the discrepancy. 

Immobilization in a sol-gel matrix accelerated the propanolysis of N-acetyl-i-phenylalanine ethyl ester in cyclohexane for several serine proteases compared to the non-immobilized lyophilized enzymes 31-fold for Subtilisin Carlsberg, 43-fold for a-chymotrypsin, and 437-fold for trypsin (van Unen, 2001). The activity yield upon immobilization was 90% (a-chymotrypsin). The rate enhancement effect of immobilization on the enzyme activity is highest in hydrophobic solvents. 

To demonstrate the synthetic application of this methodology, the authors subsequently demonstrated its use for the preparative kinetic resolutions of a series of 2° alcohols, Table 24, whereby 20 ml solutions of each racemic alcohol were passed through the bioreactor (3.3 h) and found to afford analogous results to those obtained during the initial optimization experiments. The authors successfully demonstrated the use of immobilized and lyophilized enzymes within a continuous flow reactor, presenting a synthetically viable approach to the kinetic resolution of racemic alcohols. 

Dissolve lyophilized enzyme to 1000 units/ml in concentrated enzyme storage buffer. Divide into 20-pi aliquots and store at —20°C. 

Lyophilized enzymes usually contain residual water, which is not... 

U/ml inorganic pyrophosphatase from Baker s yeast (Sigma-Aldrich, St. Louis, MO, USA). Resuspend 500 U lyophilized enzyme and buffer salts in 250 pi water. Store at -80 °C in 5 pi aliquots. Store thawed aliquot on ice and discard at the end of assay day. 

The presence of crown ethers in aqueous medium before lyophilization can greatly enhance the activity of proteases (e.g., chymotrypsin, subtilisin, trypsin) for peptide synthesis in organic solvents. - The crown ether must also be rinsed away from the lyophilized enzyme before use. The activity enhancement is solvent dependent. It is speculated that crown ethers accelerate enzymatic rates either by preventing a salt bridge from forming in the enzyme s secondary structure or inducing microscopic changes in the structure of the solid phase. ... 

In addition to PEG, often employed as a lyoprotectant, other polymers, when present in the aqueous mixture, can yield more active lyophilized enzymes. -... 

Immobilization, dehned as the physical confinement or localization of an enzyme into a specihc micro-environment, has been a very common approach to prepare enzymes for aqueous as well as nonaqueous applications. For nonaqueous enzymol-ogy, immobilization improves storage and thermal stability, facilitates enzyme recovery, and enhances enzyme dispersion. In addition, immobilized enzymes are readily incorporated in packed bed bioreactors, allowing for continuous operation of reactions. Moreover, lyophilized enzyme powders often aggregate and attach to reactor walls, particularly when the water activity is moderately high. The major disadvantage of immobilization is low activity, induced by pore diffusion mass transfer limitations and by alteration of protein stmcture. For enzymes in nonaqueous media, the following broad categories of immobilization exist ... 

However, becanse lyophilized enzymes contain altered stracture relative to aque-ons phase enzymes, protein structures obtained from molecnlar modehng with the Brookhaven database (aqneons-derived) are not relevant. For cases involving lyophilized enzymes, and when the desolvated portion of snbstrates cannot be determined, the same research gronp discovered that the following eqnation, similar to that given above, was effective. ... 

Acetylcholinesterase (AChE) The lyophilized enzyme from electric eel (Sigma) was dissolved in 0.05M phosphate buffer (pH-7.4) at a concentration of 20 pg/mL. Acetylthiocholine iodide was used as substrate at a final concentration of 5x10 4M in buffer. 5,5 -Dithiobis-(2-nitrobenzoic acid) (DTNB) at a final concentration of 3.8xlO 2M was used to monitor the released thiocholine according to a published procedure (41) with slight modification. Acetone was used as a solvent for the inhibitors. 

A simple way of applying enzymes in a non-aqueous reaction is through addition of lyophilized enzyme powder. With a hydrophobic solvent, this will create an enzyme suspension and, if other conditions (substrate, temperature, etc.) are acceptable, it will probably work. For a quick screening of a range of enzymes it may even be the most optimal setup. It is, however, well known that many enzymes lose activity upon lyophilization (which to some degree can be prevented by the use of lyoprotectants). Another concern is that enzyme dust is potentially allergenic if inhaled. 

Crystals, uv max 280 nm (e 66,300). Optimum pH 7.0-8.5 stable at pH 6.0-9.0. The refrigerated lyophilized enzyme is stable for months frozen enzyme soln can be kept for weeks without significant loss of activity. Not deactivated at 65% but loses half of its activity upon heating at 80" for I hr. 

Improving Activity of Salt-Lyophilized Enzymes in Organic Media... 

Native and modified SC were lyophilized with 0, 50, 75, and/or 99% salt concentration (in the lyophilized preparation). The salt mixture used contained 75% sodium bicarbonate and 25% sodium acetate by weight [13], with the total salt concentration at 1 M in the salt-SC solution used for lyophilization. The solution also contained 1 mg/mL potassium phosphate, pH 7.8, to buffer the lyophilized enzyme. The salt-SC solutions were lyophilized for a period of 36 h at -50°C and 8 im Hg. 

Overall, chemical modification and surfactants appear to offer moderate enhancements in some cases compared to salt-lyophilized enzymes. However, these enhancements are small compared to the original advantages of salt lyophilization. 

All detergents tested were inhibitory, and the best results were obtained by mixing lyophilized enzyme with a benzene solution of the diglyceride, and evaporating off the solvent. 

This phenomenon is not observed using SOD which was previously treated with organic solvents or lyophilized enzyme from either source stored for three months. It was suggested, that the molecular architecture of the active center of aqueously isolated enzyme differs from that of the other species The molecular properties of the SOD s summarized in the next chapter are essentially all derived from data collected from the enzyme which was isolated employing the diloroform/ethanol method. Data from Cu Zn superoxide dismutases obtained by other isolation methods are awaited with great interest. 

---