Кемерово, Кемеровская область, Россия
Modern methods of chemical modification of enzymes conferring increased catalytic activity and stability to these molecules have been considered. The advantages of using magnetic nanoparticles for the production of stable im-mobilized enzyme preparations are presented. Chymotrypsin immobilization on Fe3O4 nanoparticles modified with amino groups has been found to result in the incorporation of 88% of the enzyme into the solid phase. The change of the optimal pH and temperature ranges and an increase of stability of the immobilized chymotrypsin relatively to the respective characteristics of the native enzyme have been demonstrated.
catalytic activity, magnetic nanoparticles, chymotrypsin, glutaraldehyde method, immobilization
INTRODUCTION
The possibilities of application of enzymes, especial-ly in medicine and food processing, have expanded significantly due to the recent advances in enzymology. This is due to the obvious advantages of enzymes over chemical catalysts, namely, selectivity and stereospeci-ficity of action, the ability to attain high substrate con-version rates under relatively mild technological condi-tions, and safety for the environment and humans [1, 2].
Most enzymes used in food processing are present in food and are ingested with fresh fruit and vegetables, nuts, milk, and fermented and canned foods. The search for new applications of enzymes in various fields of biotechnology is currently going on. The main areas of research include modification of the properties of individual enzymes in order to increase their activity and reduce the cost of the end products, screening of novel enzyme-producing microorganisms, generation of novel recombinant enzymes with desired properties, the use of enzymatic reactions for the production of valuable food ingredients and bioactive substances, and the development of enzyme-based nanotechnology procedures for food processing [2].
The modern methods of enzyme modification confer increased resistance to a variety of chemicals and inhibitors, as well as to pH and temperature effects, to these molecules and allow for alteration of the pH optimum, substrate specificity, and binding properties of the enzymes. Moreover, the catalytic properties of enzymes and the preference of these biocatalysts towards certain metal cofactors can be regulated by modification [2].
Chemical modification of enzymes is among the most widely used methods [3, 4]. The chemical modifi-cation procedures must meet a number of requirements. Firstly, the method should employ non-hazardous chemical reagents, especially in case of enzymes subsequently used in food industry. Secondly, harsh conditions of modification leading to enzyme deterioration should be avoided. Thirdly, separation of the modified enzymes from the reaction medium by relatively simple and inexpensive methods should be possible. Finally, the use of the modified enzymes should be cost-effective [4].
The use of non-polar reaction media is an example of chemical modification of enzymes [5]. The resulting reduction of water activity in the reaction system leads to substantial changes of the properties of enzymes, namely, the reaction is shifted towards synthesis, the thermal stability and the storage stability of the enzyme increase, the enzyme acquires an ability to catalyze novel reactions not occurring in an aqueous environ-ment and retains activity in organic solvents at a tem-perature above 100°C. This method of chemical modification is applicable for such enzymes as lipase, chymotrypsin, trypsin, subtilisin, thermolysin, polyphenol oxidase, glucoamylase, papain, and chymosin.
Research on biological methods of enzyme modification is an actively developing area of enzymology. Protein engineering is an especially promising approach. The methods of protein engineering based on information on the relationship between amino acid sequence, three-dimensional structure, and catalytic activity of enzymes allow for successful modification of enzymes resulting in improvement of their technological characteristics [6, 7]. Substitution of certain amino acid residues in the enzyme molecule is a widely used method.
Substitution of amino acid residues in the enzyme molecules can be used to alter the substrate specificity of these biocatalysts [8]. For example, the ratio of cello-biohydrolase activity towards soluble and insoluble substrates can be altered by replacing the external aromatic amino acid residues which bind to the end of the polysaccharide molecule and direct it into the active site. Resistance of the enzyme to high temperature and extreme pH values is achieved by replacing pairs of amino acid residues located close to each other in the tertiary structure of the enzyme in order to create additional non-covalent hydrophobic bonds, salt bridges, or covalent S-S bonds conferring higher general stability to the globular enzyme molecule.
1. Volova, T.G., Biotekhnologiya (Biotechnology), Novosibirsk: Izdatel’stvo SO RAN, 1999.
2. Biotekhnologiya (Biotechnology), Higgins, I., Best, D., and Jones, G., Eds., Moscow: Mir, 1988.
3. Eggins, B., Khimicheskie i biologicheskie sensory (Chemical sensors and biosensors), Moscow: Tekhnosfera, 2005, no. 3, pp. 25-31.
4. Gracheva, I.M., Tekhnologiya fermentnykh preparatov (Technology of enzyme preparations), Moscow: Agropromizdat, 1985.
5. Antipova, L.V., Primenenie fermentov v pererabotke vtorichnogo molochnogo syr’ya (The use of enzymes in the processing of secondary raw materials of dairy origin), Moscow: AgroNIITEIMMP, 1992.
6. Andreeva, I.N., Pantyukhin, A.V., and Sysuev, B.B. Immobilizatsiya fermentov i drugikh biologicheski aktivnykh soedinenii. Uchebnoe posobie (Immobilization of enzymes and other biologically active substances. Study guide), Pyatigorsk: Pyatigorskaya Gosudarstvennaya Farmatsevticheskaya Akademiya, 2001.
7. Berezin, I.V. Biotekhnologiya. Uchebnoe posobie dlya vuzov. T. 7. Immobilizovannye fermenty (Biotechnology. Stu-dent training manual. Vol.7. Immobilized enzymes), Moscow: Vysshaya Shkola, 1987.
8. Berezin, I.V., Klesov, A.A., and Shvyadas, V.K., Inzhenernaya enzimologiya (Engineering enzymology), Moscow: 1987.
9. Dixon, M. and Webb, E., Fermenty. Tom 1,2 (Enzymes. Vols. 1, 2), Moscow: Mir, 1982.
10. Berry, C., and Curtis, A., Functionalization of magnetic nanoparticles for applications in biomedicine, J. Phys. D. Appl. Phys., 2003, vol. 36, pp. R198-R206.
11. Gu, H., Xu, K., and Xu, C., Biofunctional magnetic nanoparticles for protein separation and pathogen detection, J. of the American Chemical Society Chem. Commun., 2006, pp. 941-949.
12. Massart, R., Preparation of aqueous magnetic liquids in alkaline and acidic media, IEEE Trans. Magn., 1981, vol. 17, no. 2, pp. 1247-1248.
