Lyophilized enzyme powder

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]. 

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 ... 

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. 

Hydrolases, especially lipases, which normally act at a water-organic interface, remain active in many organic solvents. Nonpolar solvents like hexane toluene or t-butyl methyl ether are best. Hydrolase activity in polar solvents like methanol or DMSO is poor, likely because they hydrogen bond to the protein main chain and disrupt the folded conformation. Hydrolases do not dissolve in organic solvents, but are used as lyophilized enzyme powders suspended in the organic solvent or as immobilized enzymes. 

A nitrilase enzyme (BD9570,0.1 unit/mg, Diversa) was used to desymmetrize prochiral substrate 14 to afford (R)-15. In this process, an aqueous solution of 100 mM NaH PO at pH 7.5 (510 mL) was added to lyophilized nitrilase enzyme powder (15.15 g). The mixture was stirred at 27°C (to rehydrate the lyophilized enzyme powder) for 40 min. 3-Hydroxyglutaronitrile (252.5 g) was then charged over 10 min. The mixture was stirred at 27°C for 16 h and then cooled to 2°C prior to acidification to give pH 2. Celite was charged (25 g) and the slurry filtered. The filtrate was extracted with methyl ethyl ketone. The combined methyl ethyl ketone extracts were evaporated in vacuo (15 mbar, 40°C) to yield the product 15 as a brown liquid (240.8 g, 81% yield ee 98.8%). 

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). 

Lyophilization employs sub-zero temperatures in combination with very low pressure to withdraw water from the protein solution typical values would be -80 °C and 1-3 mbar. In these conditions, as the water phase diagram reveals, water sublimes, leaving a fluffy porous enzyme powder. As typical run times are on the order of a day, lyophilization is not the method of choice for large-scale enzyme drying operations. On a laboratory scale, however, lyophilization is a very effective method. 

A powder of hpoprotein hpase (LPL) esterified an organic substrate in toluene at a rather poor reaction rate (Table 4), which was to some extent explained by adhesion of the sticky enzyme powder to the surface of the reaction vessel [7]. When polyethylene glycol (PEG) was bound covalently to LPL and this modified enzyme was dissolved in toluene, approximately 3.5 U mg of enzyme protein were assayed. After simple addition of PEG to the reaction mixture together with LPL powder, the same poor reaction rate of the enzyme powder alone was observed. On the other hand, when LPL powder was lyophilized together with PEG the resultant preparation had an activity of 1.8 U mg L In this case, the enzyme... 

In 1980s, transesterification reactions were mostly performed with dry, lyophilized, or freeze-dried enzyme powders under basically nonaqueous conditions or in the presence of minor amounts of water. Today, various immobilized... 

Preparation of crude enzyme. The fungus was cultivated in a liquid medium for 3 days by using 50-1 fermenter. The culture broth was concentrated by ultrafiltration to one-tenth of its volume after removal of mycelia, and lyophilized. This enzyme powder (1 g) contained 0.26 g protein, and showed 120, 600, and 60 units of activity on PNPG, CMC, and KC-floc, respectively. 

Vials containing lyophilized enzymes or cofactors (e.g., NADH) should be warmed to room temperature before opening. This prevents condensation of moismre on the powder, which can cause loss of activity or degradation. If the reagent is hygroscopic, one such mishandling may ruin the entire vial. 

Aprotinin is a polypeptide consisting of a chain of 58 amino acid residues, which inhibits stoichiometrically the activity of several proteolytic enzymes such as chymotrypsin, kallikrein, plasmin, and trypsin. Aprotinin is obtained from bovine tissues and purified by a suitable process. It is stored as a bulk solution or lyophilized powder. The amount of two related substances des-Ala-des-Gly-aprotinin and des-Ala-aprotinin is determined by CZE with a 100% analysis. The relative migration times are 0.98 for des-Ala-des-Gly-aprotinin and 0.99 for des-Ala-aprotinin, and the specified limits are 8.0 and 7.5%, respectively. 

Figure 13. Preparation of immobilized enzymes with different solubilities in aqueous solutions and organic solvents. Procedure A mixture of an enzyme (3 mg) and the polymer (10 mg) was incubated at pH 7.5 for 20 min. Ammonium phosphate (0.1 M, pH 7, 1 mL) was then added to react with the remaining active ester. After 20 min, the solution was ready for use, or lyophilization to give the immobilized enzyme as a powder to be used for reaction in organic solvents. Each gram of the polymer contains approximately 0.7 mmol of the active ester.

The presence of small amounts of water was found to be essential even for hydrophobic ionic liquids (284). When a-chymotrypsin (in the form of salt-free lyophilized powder) was applied for the transesterification of Ai-acetyl-L-phenyl-alanine ethyl ester with 1-propanol in the dry ionic liquids [BMIM]PFg and [OMIMJPFg, little enzymatic activity was observed. The maximum activity was observed when 0.5 vol% water was added to the ionic liquids. Supercritical CO2 was also sufficient to activate the enzyme in dry ionic liquids. The addition of water to the supercritical C02-ionic liquid system further enhanced the enzymatic activity. 

Over the years there have been many failures with LNIT, due to the absence of standardization and the presence of local adverse reactions, particularly evident with the use of aqueous extracts. These inconvenients are synthesized in little stability (some molecules adhere to the inside surface of the container), autodigestion (presence of proteolytic enzymes in the extracts), and it is impossible to add propellants that enable their vaporization. It has become possible to overcome these problems after the realization of lyophilized allergenic preparations incorporated into an inert excipient in powder (lactose) and included in a rigid gelatine capsule [10, 26],... 

Enzymes and Protein Assays. Aliquots of sonicated cells were adjusted with 0.32 M sucrose to give 10% homogenates which were they lyophilized, and stored over CaC desiccant at -20%. When needed, portions of the powder were reconstituted with water to the original concentration of about 6 to 10 mg protein per ml. 

A measure as simple as adding certain inorganic salts to aqueous enzyme solutions prior to lyophilization can result in dramatic activation of the dried powder in organic media relative to enzyme lyophilized without added salt (Ru, 1999). 

Rees DG, Hailing PJ (2001) Chemical modification probes accessibility to organic phase proteins on surfaces are more exposed than in lyophilized powders. Enzyme Microb Technol 28 282-292... 

Its utility as an enzyme label is due in part to its relatively good stability characteristics (as a lyophilized powder it may be stored for years at 4°C and as a purified aqueous solution it can retain undiminished activity for over 12 months at 4 ° C). H RP has a high specific activity and broad substrate specificity. The iron atom within the active site has a single free molecular orbital available for substrate and oxidant interaction hence, activity can be reversibly inhibited by alternative ligands such as cyanide and sulfide at concentrations of about 10- M. 

The activities of several enzymes have been studied in partially hydrated powders as a funcuon of water activity or water content. Experiments of this type are not difficult to perform. Solutions of substrate and enzyme are mixed quickly, and the mixture is immediately frozen and lyophilized, which stops the reaction and gives a stable dry powder. If appropriately high concentrations of enzyme and substrate are mixed, the powder is of the enzyme-substrate complex. The sample is rehydrated under a controlled atmosphere to give the desired final hydration level. Conditions, particularly the pH of the sample, are set such that the hydration equilibrium is substantially complete (within several hours) before appreciable enzyme reaction has taken place. The problem of defining pH in partially hydrated powders was discussed in Section II,D in connection with hydrogen-exchange measurements. The pH of a powder appears to equal the nominal pH (that of the solution from which the powder was lyophilized) above about 0.15 A. 

Likewise, the strucmre of subtilisin (pH 3.0) suspended in varying ratios of acetonitrile and water demonstrated a-helical content similar to that in the lyophilized powder (Griebenow and Klibanov, 1996). Furthermore, the rate of transesteriflcation reactions of subtilisin (pH 7.8) suspended in DMSO/acetonitrile, formamide/acetonitrile or formamide/dioxane were increased approximately 100-fold over aqueous conditions (Almarsson and Klibanov, 1996). Similar results were obtained for subtilisin (pH 7.8) in a tetrahydrofuran/1-propanol mixture (Affleck et al., 1992). These results can be attributed to the increased structural rigidity of the active conformation of the protein in the solid, and the denaturing characteristics of the solvent at the solvent-particulate interface. Preservation of this molecular memory or molecular imprint of the protein can also be used to stabilize structure and activity (Mishra et al., 1996 Rich and Dordick, 1997 Santos et al., 2001). Subtilisin was lyophilized from crown ethers, resulting in more native like structure, by FTIR, and increased enzyme activity in THF, acetonitrile and dioxane (Santos et al.,2001). 

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