Chemical Modification of Lysozyme with Dextran by Using Maillard Reaction and Evaluating the Antimicrobial Properties of the Modified Enzyme

Subject Areas : Microbiology

Mahmood Aminlari ¹ , Roghayeh Ramezani ² , sedigheh amiri ³

1 - استاد دانشگاه شیراز، دانشکده دامپزشکی، گروه علوم پایه، شیراز، ایران
2 - مربی دانشگاه شیراز، دانشکده کشاورزی، بخش علوم و صنایع غذایی، شیراز، ایران
3 - عضو هیات علمی دانشگاه آزاد اسلامی، واحد یاسوج، گروه صنایع غذایی، باشگاه پژوهشگران جوان، یاسوج، ایران

Received: 2012-04-29 Accepted : 2012-09-11 Published : 2013-12-22

Keywords: Antimicrobial effect, Conjugate, Dextran, Lysozyme,

Abstract :

Introduction: Lysozyme is a natural enzyme with positive antimicrobial activity against Gram Positive  bacteria, but its action is limited on Gram Negative bacteria, which is assumed a restriction for its uses in industry. The purpose of this research was to glycosylate lysozyme with dextran through Maillard-based reaction and to study the antimicrobial characteristics of lysozyme-dextran conjugate.Materials and Methods: Glycosylation of lysozyme with dextran was performed using a 1:5 weight ratio of protein to dextran, incubated at 60 MC for one week under the relative humidity of 79%. For evaluating conjugation of dextran to lysozyme, SDS-PAGE electrophoresis was conducted. Gel filtration chromatography with sephadox G-100 was used for separation of conjugated enzyme. Lytic activity, free amino group and antimicrobial activity of the modified enzyme were evaluated.Results: SDS-PAGE electrophoresis was used to follow the glycosylation process. Results indicated that 3.7 moles of dextran were coupled to one mole of lysozyme. The lytic activity of the conjugate was about 62% of that of the native lysozyme. Evaluation of antimicrobial activity of the lysozymedextran conjugate, indicated the effectiveness of modified enzyme against E. coli and a progressive increase in antimicrobial activity with an increase in enzyme-conjugate concentration. The antimicrobial action of lysozyme on S. aureus was not improved by conjugation with dextran as compared with that of E. coli.Conclusion: These results might increase the application of lysozyme as a natural antimicrobial ingredient in different food systems.

References:

Aminlari, M., Ramezani, R. & Jadidi, F. (2005). Effect of Maillard-based conjugation with dextran on the functional properties oflysozyme and casein. Journal of the Science of Food and Agriculture, 85, 2617-2624.

Arita, K., Babiker, E., Azakami, H. & Kato, A. (2001). Effect of chemical and genetic attachment of polysaccharide to proteins on the production of IgG and IgE. Journal of Agricultural and Food Chemistry, 49, 2030-2036.

Devlieghere, F., Vermeiren, L. & Debevere, J. (2004). New preservation technologies: possibilities and limitations. International Dairy Journal, 14, 273-285.

Delaney, R. (1980). Applied protein chemistry. Applied Science Publishers, London, pp. 233-280.

Gill, A. O. & Holley, R. A. (2003). Interactive inhibition of meat spoilage and pathogenic bacteria by lysozyme, nisin and EDTA in the presence of nitrite and sodium chloride at 24˚C. International Journal of Food Microbiology, 80, 251-259.

Gill, A. O. & Holley, R. A. (2000). Inhibition of bacterial growth on ham and bologna by lysozyme, nisin and EDTA. Food Research International, 33, 83-91.

Habeeb, A. F. S. A. (1966). Determination of free amino groups in proteins by trinitrobenzenesulfonic acid. Analytical Biochemistry, 14, 328-336.

Hattori, M., Imamura, S. & Nagasawa, K. (1994). Functional changes of lysozyme-carboxymethyl dextran conjugate. Bioscience Biotechnology and Biochemistry, 58, 174-177.

Haynes, R., Osuga, D. T. & Feeney, R. E. (1967). Modification of amino groups in inhibitors of proteolytic enzymes. Journal of Biochemistry, 6, 541-546.

Ibrahim, H. R., Higashiguchi, S., Juneja, L. R., Kim, M. & Yamamoto, T. (1996). A structural phase of heat-denatured lysozyme with novel antimicrobial action. Journal of Agricultural and Food Chemistry, 44, 1416-1423.

 

Ibrahim, H. R., Higashiguchi, S., Koketsu, M., Juneja, L. R., Kim, M. & Yamamoto, T. (1996). Partially unfolded lysozyme at neutral PH agglutinates and kills Gram-negative and Gram-positive bacteria through membrane damage mechanism. Journal of Agricultural and Food Chemistry, 44, 3799-3806.

Ibrahim, H. R., Hatta, H., Fujiki, M., Kim, M. & Yamamoto, T. (1994). Enhanced antimicrobial action of lysozyme against Gram-negative and Gram-positive bacteria due to modification with perillaldehyde. Journal of Agricultural and Food Chemistry, 42, 1813-1817.

Imoto, T., Johnson, L. N., North, A. C. T., Philips, D. C. & Rupley, J. A. (1972). Vertebrate Lysozyme. Academic Press, New York, pp. 665-664.

Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T₄. Nature, 227, 680-685.

Lowry, P. H., Rosebrough, N. J. & Randall, R. J. (1951). Protein measurement with the folin phenol reagent. Journal of Biological Chemistry, 193, 265-275.

Naidu, A. S. (2000). Natural Food Antimicrobial Systems. CRC Press, Culinary and Hospitality Industry Publications Services, Washington, pp. 1– 4.  

Nakamura, S., Kato, A. & Kobayashi, K. (1991). New antimicrobial characteristics of Lysozyme–dextran conjujate. Journal of Agricultural and Food Chemistry, 39, 647–650.

Nakamura, S., Kobayashi, K. & Kato, A. (1994). Role of positive charge of lysozyme in the excellent emulsifying properties of Maillard-type lysozyme-polysaccharide conjugate. Journal of Agricultural and Food Chemistry, 42, 2688-2691.

Song, Y., Babiker, E. E., Usui, M., Saito, A. & Kato, A. (2002). Emulsifying properties and bactericidal action of chitosan-lysozyme conjugates. Food Research International, 35, 459-466.

Scaman, C., Nakai, S. & Aminlari, M. (2006). Effect of pH, temperature and sodium bisulfite or cyctein on the level of Maillard–based conjugation of lysozyme with dextran, galactomannan. Food Chemistry, 99, 368 –380.

Takahashi, K., Lou, X., Ishii, Y. & Hattori, M. (2000). Lysozyme–glucose stearic acid monoester conjugate formed through the Maillard reaction as an antibacterial emulsifier. Journal of Agricultural and Food Chemistry, 48, 2044-2049.

Touch, V., Hayakawa, S. & Saitoh, K. (2004). Relationship between conformational changes and antimicrobial activity of lysozyme upon reduction of its disulfide bonds. Food Chemistry, 84, 421-428.