Lyophilization, enzyme

The simplest way to prepare a biocatalyst for use in organic solvents and, at the same time, to adjust key parameters, such as pH, is its lyophilization or precipitation from aqueous solutions. These preparations, however, can undergo substrate diffusion limitations or prevent enzyme-substrate interaction because of protein-protein stacking. Enzyme lyophilization in the presence of lyoprotectants (polyethylene glycol, various sugars), ligands, and salts have often yielded preparations that are markedly more active than those obtained in the absence of additives [19]. Besides that, the addition of these ligands can also affect enzyme selectivity as follows. 

Activase 550/20 mg Thrombolytic enzymes lyophilized recombinant tPA Genentech... 

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). 

Enzyme 0.228 mM in 0.1 M phosphate buffer (pH 7.0), temperature 105 K, 100 kHz modulation frequency, 1.0 mT modulation amplitude, 2 mW power (20 dB), 0.2 s time constant, 0.10 mTls scan rate, 9.39148 GHz microwave frequency. Key 1, oxidized enzyme P as 1, but 5 X instrument sensitivity 2, reduced enzyme, sensitivity as in V 3, oxidized enzyme, lyophilized at 0 C, redissolved. Asm Agio = 1.1 4, oxidized enzyme, after treatment with Chelex 100, AisolAno = 1.0, sensitivity in 3 and 4 as in V. (Reproduced, with permission, from Ref. 18. Copyright 1979,... 

To increase dispersion or surface area of salt-enzyme preparations in organic media, a method of preparing surfactant-assisted salt enzyme nanodispersions (SSENDs) was explored. The goal was to create a fine dispersion of the salt enzyme lyophilized particles with the use of a siufactant prior to lyophilization by creation of a fine emulsion, followed by removal of solvent and water by lyophilization. Microscopic observation of the emulsion indicated a submicron dispersion of the aqueous phase in the solvent. The results of the ertzyme assay using SSENDs catalyst is shown in Table 3. This experiment was conducted in hexane to determine if such a preparation can enhance activity of the enzyme because posdyophilization addition of the surfactant did not improve the rate of the reaction. The results show that a two- to threefold higher rate can be obtained by preparation of the salt-SC using the SSENDs preparation method. 

C, partially purified dogfish enzyme, lyophilized, almost indefinitely [18]... 

Freeze drying, or lyophilization, is normally reserved for temperature-sensitive materials such as vaccines, enzymes, microorganisms, and therapeutic proteins, as it can account for a significant portion of... 

In these systems, solid enzyme preparations (e.g. lyophilized or immobilized on a support) are suspended in an organic solvent in the presence of enough aqueous buffers to ensure catalytic activity. Although the amount of water added to the solvent (as a rule of thumb <5% v/v) may exceed its solubility in that solvent, a visible discrete aqueous phase is not apparent because part of it is adsorbed by the enzyme. Therefore, the two phases involved in an organic solvent system are a liquid (bulk organic solvent and reagents dissolved in it) and a solid (hydrated enzyme particles). 

However, in most cases enzymes show lower activity in organic media than in water. This behavior has been ascribed to different causes such as diffusional limitations, high saturating substrate concentrations, restricted protein flexibility, low stabilization of the enzyme-substrate intermediate, partial enzyme denaturation by lyophilization that becomes irreversible in anhydrous organic media, and, last but not least, nonoptimal hydration of the biocatalyst [12d]. Numerous methods have been developed to activate enzymes for optimal use in organic media [13]. 

Basically, there are three ways to tune enzyme enantioselectivity by means of additives (i) the additives are placed in the reaction medium together with the organic solvent, the enzyme, and the reagents (ii) the additives are co-lyophilized with the biocatalyst before use in the organic solvent (iii) the additives are complexed with the substrates before their transformation in the organic medium. 

The rotors can be preloaded with lyophilized reagents, which can be dynamically dissolved by the addition of buffer to the spinning rotor. Multiple samples can then be introduced into each of the radial cuvettes, or a single sample can be dynamically apportioned between the multiple cuvettes, each of which contain reagents for a different enzyme reaction. Consequently, multiple samples can be monitored for the same enzyme activity, or several different enzyme activities can be measured for the same sample. The very fast data reduction offered by the online computer provides the operator with printed results as soon as the analysis is complete. This approach provides highly precise data (Table II). 

Enzyme Reference Serums. Several companies sell lyophilized or stabilized reference serums for the calibration of instruments and for quality control. The label values given for the enzymatic activity of these serums should never be taken at face value, as at times they may be quite erroneous (19,33). Also, these values should only be used for the assay with which they were standardized, as interconversion of activity from one method to another for the same enzyme may often lead to marked errors. For instance, it is not recommended that alkaline phosphatase expressed in Bodansky units be multiplied by a factor to convert it to the units of the Ring-Armstrong method, or any other method for that matter. 

Endopolygalacturonases PGI and PGII isolated from a recombinant Aspergillus, niger and exopolygalacturonase (PGX) isolated from A. tubingensis are described elsewhere in this volume (see Benen et al. and Kester et al., respectively). For NMR spectroscopy the enzymes were lyophilized three times from DjO. 

Enzymes are suspended in hydrated microemulsion surrounded by a monolayer of surfactant molecules dispersed in an apolar solvent [53-60,135] [Fig. 1(b)]. Micelles ( 2 nm sphere) are formed when lyophilized or aqueous preparation of enzymes are introduced with stirring or shaking into a solution of synthetic or natural surfactant in an organic solvent. 

In another approach, the alcohol moiety, formed by an enzymatic hydrolysis of an ester, can act as a nucleophile. In their synthesis of pityol (8-37a), a pheromone of the elm bark beetle, Faber and coworkers [17] used an enzyme-triggered reaction of the diastereomeric mixture of ( )-epoxy ester 8-35 employing an immobilized enzyme preparation (Novo SP 409) or whole lyophilized cells of Rhodococcus erythro-polis NCIMB 11540 (Scheme 8.9). As an intermediate, the enantiopure alcohol 8-36 is formed via kinetic resolution as a mixture ofdiastereomers, which leads to the diastereomeric THF derivatives pityol (8-37a) and 8-37b as a separable mixture with a... 

Another very recent development in the field of enzymatic domino reactions is a biocatalytic hydrogen-transfer reduction of halo ketones into enantiopure epoxides, which has been developed by Faber, Bornscheuer and Kroutil. Interestingly, the reaction was carried out with whole lyophilized microbial cells at pH ca. 13. Investigations using isolated enzymes were not successful, as they lost their activity under these conditions [26]. 

Each enzyme has a working name, a specific name in relation to the enzyme action and a code of four numbers the first indicates the type of catalysed reaction the second and third, the sub- and sub-subclass of reaction and the fourth indentifies the enzyme [18]. In all relevant studies, it is necessary to state the source of the enzyme, the physical state of drying (lyophilized or air-dried), the purity and the catalytic activity. The main parameter, from an analytical viewpoint is the catalytic activity which is expressed in the enzyme Unit (U) or in katal. One U corresponds to the amount of enzyme that catalyzes the conversion of one micromole of substrate per minute whereas one katal (SI unit) is the amount of enzyme that converts 1 mole of substrate per second. The activity of the enzyme toward a specific reaction is evaluated by the rate of the catalytic reaction using the Michaelis-Menten equation V0 = Vmax[S]/([S] + kM) where V0 is the initial rate of the reaction, defined as the activity Vmax is the maximum rate, [S] the concentration of substrate and KM the Michaelis constant which give the relative enzyme-substrate affinity. 

Purified preparations of calf intestinal AP maintained in solution are usually stored in the presence of a stabilizer, which is typically 3 M NaCl. The enzyme also may be lyophilized, but it may experience activity loss with each freeze-thaw cycle. AP is not stable under acidic conditions. Lowering the pH of an AP solution to 4.5 reversibly inhibits the enzyme. It is recommended that all handling, storage, and use of AP be done under conditions >pH 7.0 to maintain the highest possible catalytic activity. 

Enzymes that contain carbohydrate, such as HRP or GO, may be oxidized with periodate to create reactive derivatives that subsequently can be used to label antibodies or other targeting molecules at their amine groups. The aldehyde-HRP intermediate may be stored for extended periods in a frozen or lyophilized state without loss of activity (either enzymatic or coupling potential). Avoid, however, storage in a liquid state, since polymerization may occur—resulting in precipitation and loss of activity. 

In order to preserve, as much as possible, the phenolic content in fruit and vegetable samples, the literature proposed the application of cold temperatures, even reaching to freezing, when lyophilization is the objective. These procedures also could inactivate the enzymes. The freeze-drying is largely the main preservation technique used in the studies related to the identification and quantification of the phenolic compounds of fruit... 

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. 

---