Thermochemical modification of lysozyme enables it to retain its oligomers, showing a wide spectrum of increased antibacterial activities. It also results in significant loss of hydrolytic activity of the enzyme. The objective of this study is to analyse the protective effect of resorcinol on the hydrolytic activity of modified lysozyme using a high temperature method and the oligomerization degree in the obtained preparations. Resorcinol significantly affects the enzyme’s hydrolytic activity. Samples containing resorcinol, without oxidizing agent, modified at 90 °C retained 75 % enzymatic activity of unmodified monomer, obtaining the final value of about 16 000 U/mg. The amount of lysozyme oligomers increased slightly in the samples produced under such conditions.Kemijskom modifikacijom lizozima pri povišenoj temperaturi nastaje nova forma enzima s istim udjelom oligomera, no izraženije antibakterijske aktivnosti. Pritom dolazi do značajnog gubitka hidrolitičke aktivnosti enzima. Svrha je ovoga rada bila ispitati hidrolitičku aktivnost modificiranog lizozima pri povišenoj temperaturi uz dodatatak rezorcinola kao zaštitnog agensa, te odrediti stupanj oligomerizacije dobivenih modifikacija. Rezorcinol je bitno utjecao na hidrolitičku aktivnost enzima. Uzorci koji su sadržavali rezorcinol, ali ne i oksidirajuće sredstvo, modificirani pri 90 °C zadržali su 75 % enzimske aktivnosti nemodificiranog monomera, odnosno 16 000 jedinica/mg. Količina je oligomera u lizozomu neznatno porasla u uzorcima proizvedenim pri tim uvjetima

Grzegorz Lesnierowski

Jerzy Stangierski

Renata Cegielska-Radziejewska

Robert Borowiak

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ISSN 1330-9862 scientific note(FTB-3080)Resorcinol as Protective Agent in ThermochemicalModification of LysozymeGrzegorz Lesnierowski, Robert Borowiak*, Renata Cegielska-Radziejewska andJerzy StangierskiDepartment of Food Quality Management, Faculty of Food Science and Nutrition,Poznan University of Life Sciences, Wojska Polskiego 31, PL-60-624 Poznan, PolandReceived: April 27, 2012Accepted: January 9, 2013SummaryThermochemical modification of lysozyme enables it to retain its oligomers, showing awide spectrum of increased antibacterial activities. It also results in significant loss of hy-drolytic activity of the enzyme. The objective of this study is to analyse the protective ef-fect of resorcinol on the hydrolytic activity of modified lysozyme using a high temperaturemethod and the oligomerization degree in the obtained preparations. Resorcinol signifi-cantly affects the enzyme’s hydrolytic activity. Samples containing resorcinol, without oxi-dizing agent, modified at 90 °C retained 75 % enzymatic activity of unmodified monomer,obtaining the final value of about 16 000 U/mg. The amount of lysozyme oligomers in-creased slightly in the samples produced under such conditions.Key words: lysozyme, resorcinol, modification, oligomers, hydrolytic activityIntroductionLysozyme (N-acetylmuramide glycanhydrolase, EC3.2.1.17) is a small hydrolytic enzyme, which functionsby breaking specific bonds between polysaccharides thatform the cellular wall of bacteria. Its catalytic propertiesallow a disintegration of the b-1,4-glycosidic bond be-tween the N-acetylmuramic acid and N-acetylglucosa-mine in the structure of the polysaccharide-peptide com-plex. The antibacterial properties of lysozyme are highlyinteresting for its practical applications. The enzyme iso-lated from egg white is currently widely used in foodindustry as a biopreservative of food products and alsoin medicine and veterinary medicine in treating numer-ous diseases of bacterial and viral origin (1).Naturally occurring lysozyme is mainly monomericshowing biocidal properties against Gram-positive bac-teria. It was observed that under certain environmentalconditions, the enzyme is able to form a dimer as wellas higher oligomers, while at the same time obtainingnew, valuable properties. The oligomeric form of lyso-zyme is characterized by increased antimicrobial activ-ity, e.g. against Gram-negative bacteria and other strainsthat were so far resistant to the monomeric form. Theseproperties provide for significantly broader and morepractical application of the modified enzyme (2–6).Lysozyme oligomers exhibit a new, but not com-pletely defined action. Recent reports suggest that thisunique antimicrobial activity of unfolded lysozyme is at-tributed to membrane binding and the subsequent per-turbation of its functions (7,8). Probably, the interactionof lysozyme oligomers with the membrane of Gram-ne-gative bacteria is caused by increased surface hydropho-bicity of the modified enzyme.There are some solutions to increase the oligomeri-zation degree of enzymes, including e.g. aldehyde-dex-tran intermolecular cross-linking (9,10). Among the knownmodifications, lysozyme oligomerization was obtained asa result of applying the high temperature method usingoxidants as an additional modifier besides temperature,which we described earlier (3,4,11). However, this modi-fication resulted in a significant decrease of the enzyme410 G. LESNIEROWSKI et al.: Protective Effect of Resorcinol on Lysozyme, Food Technol. Biotechnol. 51 (3) 410–413 (2013)*Corresponding author; Phone: ++48 848 7322; Fax: ++48 61 848 7512; E-mail: boro@up.poznan.plhydrolytic activity in the obtained preparations, com-pared to the monomeric form not subjected to the pro-cess.There is a paper showing how a proper immobiliza-tion may greatly improve the enzyme features (12). It maybe used to improve enzyme performance by improvingsome enzyme limitations, i.e. enzyme purity, stability,specificity, selectivity, or activity. A recent study showedthat some phenols, particularly alkylhydroxybenzenes likeresorcinol, are good regulators of enzyme activity (13,14).It was proved that these compounds, due to their chemi-cal structure and type of interactions with the proteinmolecule, alter the enzymatic activity of lysozyme, aswell as the efficiency of hydrolysis of peptidoglycan, asubstrate of the enzyme. This short communication pre-sents first results and observations of extensive researchabout resorcinol as a protective substance used in ther-mal modification of lysozyme.Materials and MethodsThe experimental material was lysozyme monomerisolated from chicken egg white of hydrolytic activity of21 252 U/mg produced by the Belovo company (Bas-togne, Belgium). For modification, lysozyme was pre-pared as a 5 % (by mass per volume) water solution atpH=4.0±0.2. The modification was carried out for 30 minat 90 and 95 °C in the Syncore Analyst reactor (Büchi,Flawil, Switzerland) with the addition of: 0.03 % (by massper volume) of resorcinol and 2.0 % of H2O2 (by volume)(both produced by Sigma-Aldrich, St. Louis, MO, USA)(sample no. 2), 0.03 % (by mass per volume) of resor-cinol (sample no. 3), or without any additives (sampleno. 1). Then the obtained preparations were freeze-dried(GT3 Leybold-Heraeus lyophilizer, Cologne, Germany)and subjected to analytical examination.Lysozyme hydrolytic activity was determined withthe use of spectrophotometric method, the principle ofwhich is based on the phenomenon of bacterial cell walllysis by the enzyme (15,16). The lytic activity of lyso-zyme was determined by monitoring the decrease in tur-bidity of a suspension of Micrococcus lysodeikticus (Sigma--Aldrich) cells at 450 nm. The activity was presented asthe rate of absorbance decrease per minute (DA450 nm/min).The lysozyme polymeric forms in the preparationsafter modification were determined by electrophoreticanalysis in polyacrylamide gel using the SE-600 appara-tus (Hoefer Scientific Instruments, Holliston, MA, USA).Polyacrylamide gel electrophoresis was carried out ac-cording to Laemmli (17) and Lesnierowski (18) using 6 %stacking gels at a current of 60 mA and 12.5 % separat-ing gels at 90 mA. The gels were stained with 0.025 %Coomassie Brilliant Blue R solution. As molecular massstandards, the following were used (in kDa): lysozyme14.3 (Sigma-Aldrich), Lydium-KLP 28 (Nika Health Prod-ucts, Smigiel, Poland) and hen albumin 45 (Sigma-Ald-rich).The amounts of polymers in individual samples werecalculated with the application of the TotalLab Quantsoftware (Nonlinear Dynamics Ltd., Durham, NC, USA).The obtained results of the determination were sub-jected to statistical analysis using STATISTICA software(19).Results and DiscussionThe presented study showed that the modificationscarried out gave a new quality to the lysozyme. Theelectrophoretic analysis revealed that each preparationobtained after modification contained new forms of lyso-zyme (Fig. 1). Apart from the monomer, we found cer-tain amounts of dimer, trimer and also tetramer of theenzyme. Based on densitometric analysis, the exactamount of the individual oligomeric forms of lysozymewas determined (Tables 1 and 2).We observed that the amount of obtained oligomersis dependent on the temperature of the enzyme modifi-cation process. Without the use of chemical additives,411G. LESNIEROWSKI et al.: Protective Effect of Resorcinol on Lysozyme, Food Technol. Biotechnol. 51 (3) 410–413 (2013)Fig. 1. Electrophoretic image of lysozyme samples modified ther-mally at 90 °C (1–3a) and 95 °C (1–3b) using: 1=no additives,2=resorcinol and oxidant (H2O2), 3=resorcinolTable 1. Average content of lysozyme oligomeric forms and hydrolytic activity of preparations obtained at the temperature of 90 °CSamplem(resorcinol)V(water)%m(H2O2)V(water)%m(dimer)V(water)%m(trimer)V(water)%m(tetramer)V(water)%m(oligomer)V(water)%HydrolyticactivityU/mgHydrolyticactivity decrease*%1a 0.00 0.0 27.5a 27.8c 8.2a 63.6a 7740a 63.6c2a 0.03 2.0 31.4b 24.3b 15.9c 71.6c 10290b 51.6b3a 0.03 0.0 31.0b 19.7a 15.4b 66.1b 15910c 25.1a*in relation to lysozyme monomer (21 252 U/mg)Different letters in columns denote significant differences of the mean values at p£0.05V(water)=100 mLmore than 63 % of oligomers were obtained at 90 °C andapprox. 67.5 % at 95 °C. Through the use of additionalchemicals, we increased the efficiency of resorcinol by2–3 % and even up to 9 % in the case of simultaneoususe of resorcinol and H2O2 (sample 2b, temperature95 °C). This preparation contained the highest amount ofdimer (33 %), which was the main oligomeric form ob-tained after the modification. According to previous stud-ies, this form of lysozyme is the most important in shap-ing a new, much stronger antibacterial activity of modifiedlysozyme (1,2,5).As a result of the modifications carried out, we alsoobserved a significant decrease in the hydrolytic activityof the obtained samples. The mean values of these activi-ties and percentage reduction compared to the unmodifiedmonomer are presented in Tables 1 and 2. The observedchanges were probably caused by radical modificationfactors such as high temperature and oxidizing sub-stance, which work destructively on the enzyme’s activecentre.Modification temperatures of 90 and 95 °C led to thedecrease of the enzyme’s hydrolytic activity by 64 and77 %. However, the addition of resorcinol resulted in thestabilization process and then a marked decline in thedestruction of hydrolytic activity was observed. Lyso-zyme modified with resorcinol at 90 and 95 °C showed25–30 % higher activity compared to the enzyme modi-fied without this additive. The protective properties ofresorcinol were also very significant when it was usedtogether with an oxidizing agent. Under these condi-tions, a greater number of oligomers was obtained andhydrolytic activity still remained high (Tables 1 and 2).Comparing the results of the research so far, wefound that the best conditions for the modification oflysozyme were when the process was carried out at90 °C with the addition of resorcinol as a protectiveagent and an oxidizer as a substance supporting theoligomerization process. There is a need for further re-search on the optimization of the process, especially de-termining the optimal dose of resorcinol, modificationparameters and other substances from the group of phe-nols, which may be even more effective. It is also neces-sary to assess the physicochemical properties of the pro-duced oligomers, the antibacterial spectrum and thestrength of their actions. Such studies are currently be-ing conducted in our laboratory.ConclusionsIn the light of the findings concerning the protectionof lysozyme during its thermal modification, we foundthat resorcinol, an organic compound from the group ofphenols, exhibits good protective properties. It both pro-tects the hydrolytic activity of lysozyme and helps in itsoligomerization. Further studies, especially those con-cerning optimization of enzyme modification processand finding other substances that could protect lyso-zyme during thermal modification, need to be conduct-ed. And finally, it is essential to evaluate the physico-chemical properties of the produced preparation.References1. G. Lesnierowski, J. Kijowski: Lysozyme. In: Bioactive EggCompounds, R. Huopalathi, R. Lopez, M. Anton, R. Schade(Eds.), Springer-Verlag, Berlin, Germany (2007) pp. 33–42.2. H.R. Ibrahim, S. Higashiguchi, L.R. Juneja, M. Kim, T. Ya-mamoto, A structural phase of heat-denatured lysozymewith novel antimicrobal action, J. Agric. Food Chem. 44(1996) 1416–1423.3. G. Lesnierowski, R. Cegielska-Radziejewska, J. Kijowski,Antibacterial activity of thermally modified lysozyme,Electronic Journal of Polish Agricultural Universities, 2 (2001)1–9.4. G. Lesnierowski, R. Cegielska-Radziejewska, J. Kijowski,Thermally and chemical-thermally modified lysozyme andits bacteriostatic activity, World’s Poultry Sci. J. 60 (2004)303–310.5. R. Cegielska-Radziejewska, G. Lesnierowski, J. Kijowski,Properties and application of egg white lysozyme and itsmodified preparations – A review, Pol. J. Food Nutr. Sci. 58(2008) 5–10.6. R. Cegielska-Radziejewska, G. Lesnierowski, J. Kijowski,Antibacterial activity of hen egg white lysozyme modifiedby thermochemical technique, Eur. Food Res. Technol. 228(2009) 841–845.7. H.R. Ibrahim, On the novel catalytically-independent anti-microbial function of hen egg-white lysozyme: A con-formation-dependent activity, Nahrung, 42 (1998) 187–193.8. H.R. Ibrahim, T. Aoki, A. Pellegrini, Strategies for newantimicrobial proteins and peptides: Lysozyme and apro-tinin as model molecules, Curr. Pharm. Des. 8 (2002) 671–693.9. M. Fuentes, C. Mateo, B.C.C. Pessela, J.M. Guisán, R. Fer-nandez-Lafuente, Purification, stabilization, and concen-tration of very weak protein-protein complexes: Shifting412 G. LESNIEROWSKI et al.: Protective Effect of Resorcinol on Lysozyme, Food Technol. Biotechnol. 51 (3) 410–413 (2013)Table 2. Average content of lysozyme oligomeric forms and hydrolytic activity of preparations obtained at the temperature of 95 °CSamplem(resorcinol)V(water)%m(H2O2)V(water)%m(dimer)V(water)%m(trimer)V(water)%m(tetramer)V(water)%m(oligomer)V(water)%HydrolyticactivityU/mgHydrolyticactivity decrease*%1b 0.00 0.0 29.7a 28.6b 9.1a 67.4a 4960a 76.7c2b 0.03 2.0 32.9c 27.3a 10.4b 76.6c 9690b 54.4b3b 0.03 0.0 31.6b 27.1a 10.6b 69.3b 14600c 31.3a*in relation to lysozyme monomer (21 252 U/mg)Different letters in columns denote significant differences of the mean values at p£0.05V(water)=100 mLthe association equilibrium via complex selective adsorp-tion on lowly activated supports, Proteomics, 5 (2005)4062–4069.10. M. Fuentes, R.L. Segura, O. Abian, L. Betancor, A. Hidal-go, C. Mateo et al., Determination of protein-protein inter-actions through aldehyde-dextran intermolecular cross--linking, Proteomics, 4 (2004) 2602–2607.11. G. Lesnierowski, R. Borowiak, High-temperature modifica-tion of lysozyme, Acta Sci. Pol. Biotechnol. 9 (2010) 23–32(in Polish).12. C. Garcia-Galan, Á. Berenguer-Murcia, R. Fernandez-Lafu-ente, R.C. Rodrigues, Potential of different enzyme immo-bilization strategies to improve enzyme performance, Adv.Synth. Catal. 353 (2011) 2885–2904.13. Yu.A. Nikolaev, N.G. Loiko, I.Yu. Stepanenko, E.F. Shanen-ko, E.I. Martirosova, V.K. Plakunov et al., Changes in phys-icochemical properties of proteins, caused by modificationwith alkylhydroxybenzenes, Appl. Biochem. Microbiol. 44(2008) 143–150.14. A.S. Petrovskii, D.G. Deryabin, N.G. Loiko, N.A. Mikhai-lenko, T.G. Kobzeva, P.A. Kanaev et al., Regulation of thefunctional activity of lysozyme by alkylhydroxybenzenes,Microbiology, 78 (2009) 144–153.15. R.M. Parry, R.C. Chandan, K.M. Shahani, A rapid and sen-sitive assay of muramidase, Proc. Soc. Exp. Biol. Med. 119(1965) 384–386.16. G. Lesnierowski, J. Kijowski, Methods of investigating en-zymatic activity and quantitative determination of lyso-zyme from hen egg white, Przem. Spoz. 12 (1995) 476–479(in Polish).17. U.K. Laemmli, Cleavage of structural proteins during as-sembly of head of bacteriophage T4, Nature, 227 (1970)680–687.18. G. Lesnierowski, Physicochemical methods of modifica-tion and measurement of lysozyme activity, PostdoctoralThesis, Poznan University of Life Sciences, Poland (2007)(in Polish).19. STATISTICA (Data Analysis Software System), v. 10, Stat-Soft, Inc, Tulsa, OK, USA (2010) (www.statsoft.com).413G. LESNIEROWSKI et al.: Protective Effect of Resorcinol on Lysozyme, Food Technol. Biotechnol. 51 (3) 410–413 (2013)

Zaštitni učinak rezorcinola pri kemijskoj modifikaciji lizozima izloženog visokoj temperaturi

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