Enzymatic activity of lysozyme

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... 

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

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. 

At molecular level, the manifestations of the biological activity appear in specific biochemical reactions, conformational behavior, and dynamical properties of biomolecules. Experimental studies of various partially hydrated enzymatic proteins show that their activity accelerates rapidly at some critical hydration levels. Onset of the enzymatic activity of urease occurs at 0.15 g/g [469]. In the presence of chymotrypsin, the acylation reaction is undetectable at hydrations /i< 0.12 g/g, but its rate grows sharply above this critical hydration level [470]. The rate of enzymatic activity of glucose-6-phosphate dehydrogenase, hexoki-nase, and fumarase becomes detectable and start to increase sharply at hK 0.20 g/g, whereas this critical hydration is about 0.15 g/g for phosphoglucose isomerase [471]. Enzymatic activity of lysozyme can be detected only when hydration level achieves h 0.20 g/g [472, 473] (see Fig. 92). 

Figure 92 The rate a of the enzymatic activity of lysozyme at various hydration levels [473].

F. Pizzitutti, F. Bruni, Glassy dynamics and enzymatic activity of lysozyme, Phys. Rev. E 64 (2001) 052905. 

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. 

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. 

A number of modification products were isolated by ion-exchange chromatography for a mixture of 7V-carboxymethylated derivatives (CM derivatives) of lysozyme, produced by treatment of the protein with mono-iodoacetic acid. In each CM derivative the position of the modified amino-acid residues was established. Possible causes of such selectivity of the process of carboxymethyla-tion are discussed. It was also shown that salt activation of CM derivatives of lysozyme proceeds more readily, the higher the degree of their modification. As a result of this, the enzymatic activity of CM derivatives of lysozyme is higher than that of the native enzyme, which is correlated with their increased affinity for the substrate (chitin). 

Various modification products have been isolated by chromatography on Amberlite CG-50 of a mixture of A-carboxymethylated derivatives of lysozyme. In each carboxymethyl derivative the position of the modified amino-acid was established. It was found that L-histidine-15, L-lysines-1, -33, -96, or -97 are blocked, whereas L-lysines-13 and -116 remain unaffected. Possible causes of this selectivity were discussed. Salt activation of carboxy-methyl derivatives of lysozyme proceeds more readily the higher the degree of modification, and the enzymatic activity of carboxymethyl derivatives of lysozyme is higher than that of the native enzyme owing to their increased affinity for substrate (chitin). 

Vertegel, A.A., Siegel, R.W., Dordick, J.S., 2004. Silica nanoparticle size influences the structure and enzymatic activity of adsorbed lysozyme. Langmuir 20, 6800—6807. 

Based on these results, Dordick et al. [78] did another study to investigate how the sizes of the silica nanoparticles infiuence the structure and enzymatic activity of adsorbed lysozyme. The same CD technique was used to observe the protein structure, and calorimetric activity assays were used to monitor the enzyme activity after adsorption. The results from the two analysis techniques were then used to... 

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. 

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. 

Growth conditions in deep-well microtiter plates were optimized with respect to optimal expression of active enzymes (Fig. 2.2.1.1). The best results were obtained with an expression time of 20 h at 37 °C (Fig. 2.2.1.1, lanes 7-9). Subsequently, E. coli cells were enzymatically disrupted by lysozyme treatment, and the carboligase activity was monitored by a modified tetrazolium salt color assay [16], This color assay is based on the reduction of the 2,3,5-triphenyltetrazolium chloride (TTC) 13 to the corresponding formazan 15, which has an intense red color (Fig. 2.2.1.2A). Before screening ofa BFD variant library, substrates and products were tested in the color assay. Neither substrate, benzaldehyde 4 nor dimethoxy-acetaldehyde 8, reduced TTC 13 however, the product 2-hydroxy-3,3-dimethoxy-propiophenone 10 already caused color formation at low concentrations of 2.5-10 mM (Fig. 2.2.1.2B). Benzoin 12 as the product also gave a color change at a similar concentration (data not shown). 

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. 

---