Lysozyme enzymatic activity

FIGURE 2.16 pH versus enzymatic activity. The activity of enzymes is very sensitive to pH. The pH optimum of an enzyme is one of its most important characteristics. Pepsin is a protein-digesting enzyme active in the gastric fluid. Trypsin is also a proteolytic enzyme, but it acts in the more alkaline milieu of the small intestine. Lysozyme digests the cell walls of bacteria it is found in tears. 

Transition-metal phosphorus trichalcogenides such as MnPS3 are able to intercalate amino acids and peptides by ion exchange. In this way, increases in the basal spacing of 0.7 and 3-4 nm are observed for the intercalation of poly-L-lysine and lysozyme, respectively [224]. Interestingly, the enzymatic activity of the immobilized protein has been detected, suggesting that the enzyme is protected against denaturation. 

Lower and coworkers [199] have investigated the adsorption of T4 lysozyme on colloidal silica. It was observed that the enzymatic activity decreased upon adsorption due to the differences in adsorbed enzyme structure and orientation as well as the electrostatic effects. 

Imidazole moiety in His plays an important role in enzymatic activity in these enzymes as an electron donor. Besides, in the case of ribonuclease A, the imidazole moiety participates directly in catalysis, and in the case of lysozyme, -COOH moiety participates in lieu of the imidazole and cooperates with -COO-. 

Competitive Inhibition Enzyme Assays. Estimates of antibody-lysozyme dissociation constants can be obtained by taking advantage of the fact that most monoclonal antibodies efficiently inhibit enzymatic activity.3 5 The combining site of HyHEL-10, which is presented as an example, also has been demonstrated by X-ray crystallography to overlap a portion of the catalytic site of lysozyme.7 A constant concentration of lysozyme is incubated with varying amounts of antibody, and amounts of free (unbound) lysozyme molecules are estimated by the proportion of catalytic activity remaining. The assay assumes that the addition of Micrococcus lysodeikticus cell walls and concurrent dilution of the antibody-antigen mixture do not disturb the equilibrium. 

The isolation of bacterial DNA described in this experiment, patterned after the work of Marmur (1961), accomplishes these objectives. Bacterial cells are disrupted by initial treatment with the enzyme, egg-white lysozyme, which hydrolyzes the peptidoglycan that makes up the structural skeleton of the bacterial cell wall. The resultant cell walls are unable to withstand osmotic shock. Thus, the bacteria lyse in the hypotonic environment. The detergent, sodium dodecyl sulfate, (SDS, sodium do-decyl sulfate) then completes lysis by disrupting residual bacterial membranes. SDS also reduces harmful enzymatic activities (nucleases) by its ability to denature proteins. The chelating agents, citrate and EDTA (ethylenediamine tetraacetic acid), also inhibit nucleases by removing divalent cations required for nuclease activity. 

Table XI. Enzymatic Activities and Association Constants of Lysozyme and Its Derivatives (49)...

The study by Determan et al. [224] focuses on the effects of polymer degradation products on the primary, secondary, and tertiary structure of TT, OVA, and lysozyme after incubation for 0 or 20 days in the presence of ester (lactic acid and glycolic acid) and anhydride [sebacic acid and l,6-bis(p-carboxyphenoxy)hexane] monomers. The structure and antigenicity or enzymatic activity of each protein in the presence of each monomer was quantified. SDS-PAGE, circular dichroism, and fluorescence spectroscopy were used to assess/evaluate the primary, secondary, and tertiary structures of the proteins, respectively. ELISA was used to measure changes in the antigenicity of TT and OVA and a fluorescence-based assay was used to determine the enzymatic activity of lysozyme. TT toxoid was found to be the most stable in the presence of anhydride monomers, while OVA was most stable in the... 

Fig. 29. Enzymatic activity of lysozyme as a function of water content (grams of water per gram of sample), at pH 8, 9, and 10. , O, A, Measurements on powders hydrated by isopiestic equilibration ,, A, solvent added to powder. Powder samples were the 1 1 (GlcNAc)6-lysozyme complex, obtained by lyophilization. The reaction rate (no sec ) was determined by product analysis. From Rupley etcd. (1980).

Mobile bound ligand t = 7 x 10 sec Enzymatic activity of lysozyme 1/10 solution value... 

A highly sensitive, yet practical, means of lysozyme determination is that of McKenzie and White (1986), which uses the turbidimetric principle. An important feature of their method is the prolonged incubation of the reaction mixture so as to magnify traces of enzymatic activity. 

While it is clear that many proteins may be freeze-dried and reconstituted with little or no loss in activity, it is usually not obvious as to whether the freeze-dried protein is basically native in conformation or whether the solid-state conformation is distinctly nonnative, with the native and active conformation quickly forming during rehydration. The observation that lysozyme regains enzymatic activity in the solid state above about 20% water [56] demonstrates that a protein need not be in a predominantly aqueous system to maintain activity and presumably possess native structure. However, this observation does not necessarily imply that the structure is nonnative at lower water contents where the enzymatic activity disappears. The loss of activity at lower water contents [56] could simply be a consequence of greatly slowed kinetic processes (i.e., greatly restricted molecular mobility as the system passes into the glassy state). 

Enzymes such as salivary a-amylase and lysozyme, as well as bacterial glucosyltransferases immobilised in the pellicle layer, have been shown to maintain their enzymatic activities [13, 50, 53, 54],... 

Masuda, T., Ueno, Y., and Kitabatake, N. (2001). Sweetness and enzymatic activity of lysozyme. J. Agric. Food Chem. 49,4937-4941. 

The effect of "residual water" on either protein stability or enzyme activity continues to be a topic of great interest. For example, several properties of lysozyme (e.g., heat capacity, diamagnetic susceptibility (Hageman, 1988), and dielectric behavior (Bone and Pethig, 1985 Bone, 1996)) show an inflection point at the hydration limit. Detailed studies on the direct current protonic conductivity of lysozyme powders at various levels of hydration have suggested that the onset of hydration-induced protonic conduction (and quite possibly for the onset of enzymatic activity) occurs at the hydration limit. It was hypothesized that this threshold corresponds to the formation of a percolation network of absorbed water molecules on the surface of the protein (Careri et al., 1988). More recently. Smith et al., (2002) have shown that, beyond the hydration limit, the heat of interaction of water with the amorphous solid approaches the heat of condensation of water, as we have shown to be the case for amorphous sugars. 

It has been shown that the structure of the enzyme-substrate complex undergoes rearrangement in the rate-determining step of the lysozyme reaction (29). This rearrangement may require the mobility associated with completion of the water monolayer. It is also possible that a network of water molecules participates in the catalytic process. We favor the former alternative. Regardless of the explanation, it is important that not much water is needed for enzymatic activity and that only the strongly interacting sites must be filled before activity is observable. 

Figure 7. Enzymatic activity of lysozyme as a function of water content, at pH 8, 9, and 10. Open symbols measurements on powders hydrated by isopestic eqiulibra-tion. Closed symbols solvent added to powder.

We are grateful to Patricia Adams, for carrying out measurements of the enzymatic activity of lysozyme, and to Professor Walter Kauzmann for stimulation and critical discussions. This work was supported by NIH research grant GM-24760. 

K. Masaki, T. Aizawa, N. Koganesawa, T. Nimori, H. Bando, K. Kawano, and K. Nitta, Thermal stability and enzymatic activity of a smaller lysozyme from silk moth Bombyx morfj, J. Prot. Chem., 2001, 20,107-113. 

FIGURE 2.12 pH versus enzymatic activity. Pepsin, trypsin, and lysozyme all have steep pH optimum curves. Pepsin has maximum activity under very acidic conditions, as would be expected for a digestive enzyme that is found in the stomach. Lysozyme has its maximum activity near pH 5, while trypsin is most active near pH 6. 

The A. coerulea material was poorly crystalline, showing a broad peak at 20° 20, but the spectrum for the alkali-treated material showed a broad peak at 10.72° and two peaks at 18.72° and 19.98° 20, with close similarity to the spectrum of authentic chitosan. Optical microscopy showed that the alkali-treated products, stained with Saphranine or with other stains, preserved the morphology of the fungus, with flattened and empty structures [30] (Fig. 1). This work introduced the concept that an extended surface area of the carbohydrate polymer leads to enhanced performance, as amply confirmed by most recent works dealing with chitin and chitosan nanofibrils. In fact, the partially re-acetylated chitosan (degree of acetylation 0.23) is promptly depolymerized by lysozyme, papain, and lipase thanks to the ideal degree of acetylation for maximum enzymatic activity. Remarkably, the re-acetylated... 

---