Калининград, Калининградская область, Россия
Physicochemical and thermal properties of L-phenylalanine ammonia-lyase preparation were studied. Thermal gravimetric analysis of physical and chemical phenomena occurring in the enzyme upon heating was conducted. Heating curves were registered. Kinetic parameters of the evaporation process were identified. Stability of the enzyme to freezing was verified. Cryoscopic temperature of a concentrated L-phenylalanine ammonia-lyase sample was identi-fied. Chemical (dehydration, dissociation) and physical transformations accompanied by exothermic and endothermic effects were found to occur during freezing. Results of the thermal gravimetric analysis demonstrated feasibility of freeze-drying of L-phenylalanine ammonia-lyase preparation and allowed for determination of the optimum tempera-ture ranges for thermal treatment and prediction of operational parameters of the drying process.
L-phenylalanine ammonia-lyase, physicochemical and thermal properties, heating curves, cryoscopic temperature, lyophilization
INTRODUCTION
Despite the many years of production of dry biologi-cal preparations, no simple, cost-efficient, and precise enough method to calculate parameters of freeze-drying process at each stage has been proposed so far [1, 2]. Above all, this is due to the inherent complexity of the drying process, its physical and mathematical modeling, and complexity of mathematical tools to solve the differential equations describing thermodynamics of lyophilization process, especially of such complex systems as biopolymers [3, 4].
Therefore, studies aimed at determination of operat-ing conditions for lyophilization of an enzyme prepara-tion, L-phenylalanine ammonia-lyase, and development of freeze-drying technology are of topical issue [5].
L-phenylalanine ammonia-lyase catalyzes the reac-tion of reverse deamination of L-phenylalanine to trans-cinnamic acid and ammonia.
The enzyme is of interest as a therapeutic agent for phenylketonuria treatment and may be used for both direct therapy of phenylketonuria and production of food products free of phenylalanine [4, 5]. Besides the medical applications, the enzyme may be used in bio-technology for L-phenylalanine production from trans-cinnamic acid [6, 7].
The enzyme is subjected to freeze-drying to preserve its activity during storage.
Also, L-phenylalanine ammonia-lyase may be stored in glycerol solutions.
To determine operating conditions for freeze-drying of any product, including an enzyme preparation, ther-mophysical characteristics, together with physicochemi-cal properties, should be known. Thermophysical char-acteristics are necessary for determination of both ra-tional operating conditions and technological parame-ters [6, 7].
Therefore, the aim of the present work was to study physicochemical and thermophysical characteristics of an enzyme preparation of L-phenylalanine ammonia-lyase that would allow for selection of optimal freeze-drying modes.
MATERIALS AND METHODS
L-Phenylalanine ammonia-lyase preparation was the subject of the study.
To study L-phenylalanine ammonia-lyase as a sub-ject of drying, thermal gravimetric analysis of physico-chemical events occurring in the enzyme upon heating was to be performed.
Changes in weight of sample were registered in function of temperature upon thermal gravimetric analysis of L-phenylalanine ammonia-lyase preparation.
Study of thermophysical characteristics of the en-zyme preparation was performed by the first buffer method of two temperature–time intervals [7].
Then, enzyme stability to freezing was verified. Ali-quot of purified protein was diluted in 0.1 M Tris-HCl buffer, pH 8.5. Protein concentration in the sample was 0.2 mg/mL. Part of it was used to determine activity and concentration of the protein. The remaining sample was placed in a freezer at –18°С overnight. After thawing on ice, activity was determined [8, 9].
1. Gusarov, D.A., Freeze-drying of biopharmaceutical proteins. Russ. J. Biopharmaceuticals, 2010, vol. 2, no. 5, pp. 3-7.
2. Nikitin, E.E., and Zvyagin, I.V., Zamorazhivanie i vysushivanie biologicheskikh preparatov (Freezing and Drying of Biological Preparations), Moscow: Kolos, 1971.
3. Lykov, A.V., Teoriya suchki (Theory of Drying), Moscow: Energiya, 1968.
4. Babakin, B.S., and Lepikhina, O.E. Sovremennoe sostoyanie i perspektivy razvitiya vakuumnoi sublimatsyonnoi sushki (Modern state and prospectives for development of freeze-drying), Kholodil’naya tekhnika (Refrigeration Engineering), 2005, no. 11, pp. 56-59.
5. Kretov, I.T., Shakhov, S.V., Ryazanov, A.N., et al., Vybor optimal’nogo istochnika nagreva dlya vacuum-sublimatsyonnoi sushki produktov biologicheskogo proiskhozhdeniya (), Khranenie i pererabotka sel’khoz syr’a (Storage and Processing of Agricultural Raw Materials), 2001, no. 1, pp. 12-14.
6. Karpov, A.M., and Ulumiev, A.A. Sushka produktov mikrobiologicheskogo sinteza (Drying of Products of Microbi-ological Synthesis), Moscow: Kolos, 2000.
7. Baldi, C., Gasco, M., and Pattarino, F., Statistical procedures for optimizing the freeze-drying of a model drug in tret-butil alcohol-water mixtures, Eur. J. Pharm. Biopharm., 1994, vol. 40, no. 3, pp. 138-141.
8. Santivarangkna, C, Kulozik, U, and Foerst, Р. Effect of carbohydrates on the survival of Lactobacillus helveticus during vacuum drying, Lett. Appl. Microbiol., 2006, vol. 42, no. 3, pp. 271-276.
9. Abadias, M., Benabarre, A., Teixido, N., Usall, J., and Vinas, I., Effect of freeze-drying and protectants on viability of the biocontrol yeast Candida sake, Int. J. Food Microbiol., 2001, vol. 65, no. 3, pp. 173-182.
10. Champagne, C.P., Gardner, N., Brochu, E., and Beaulieu, Y., A review: the freeze-drying of lactic acid bacteria, Can. Inst. Food Sci. Technol. J., 1991, vol. 24, pp. 118-128.
