Lysozyme digestion

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. 

One of the minor by products of the isolation procedure of the disaccharide from lysozyme digests of bacterial cell walls was the tetrasaccharide /i-GIcNAc-G 4)-/i-MurNAc-G 4)-/)-GlcNAc-(l 4)-MurNAc. This structure is readily cleaved by lysozyme, and has proved to be extremely useful in other laboratories for the study of the mechanism of action of the enzyme. 

The teichoic acids of the wall and membrane act as a buffer system maintaining a concentration of Mg2 in the range of 10-15 mM, which is the optimum value for the activity of enzymes associated with membranes 31). Changes of the Mg2 + concentration in the medium have no effect on the activity of membrane-bound enzymes if the system keeps both the membrane and the cell wall closely in contact. Preparations devoid of cell walls (lysozyme digestion) exhibit a dependence of enzyme activity on the Mg2 + concentration. The membrane fragments without LTAs show a marked influence of the Mg2+ concentration on enzyme activity 31). 

Compariion of ExperiOMntal and Predicted Band Width Values for Reversed-Phase Gradient Elution of Lysozyme Digest ... 

The action of lysozyme on partially deacetylated chitin and chitin oligosaccharides has been studied.Chitin was deacetylated to an extent of about 70% of its 2-acetamido-2-deoxy-D-glucosyl residues, and lysozyme digests of the deacetylated chitin were fractionated by gel chromatography, paper chromatography, and paper electrophoresis. 

C -(2-Acetamido-2-deoxy 3-D-glucosyl)-(l 4)-iV-acetyl muramoyl-L-alanyl-D-isoglutamine (31), the dipeptide derivative of the disaccharide isolated from lysozyme digests of bacterial cell walls, has been prepared in a fourteen-step sequence from 2-acetamido-2-deoxy-D-glucose. The dipeptide disaccharide derivative (32) isolated from lysostaphin endo-l3-D-2-acetamido-2-deoxy-glucan-ase digests of cell walls has also been synthesized (Scheme 5). ... 

Fig. 2 is a modified version of Fig. 6 of a 1971 review (40) the production of hydrosoluble WSA preparations by lysozyme digestion of cell walls can only be explained by the absence of mycolic acids on some of the arabinogalactan moieties. WSA, MW 20,000 daltons contains about 15 ArajGah units, one phosphodiester bridge and 5 peptidoglycan units. The WSA unit shown here has a mol. weight of about 2.000 daltons. 

Further degradation of low molecular weight fractions obtained from lysozyme digestion of cell walls then led to the conclusion that an N-acyl muramyl dipeptide should be the minimal adjuvant active structure (small square frame of Fig. 2). And indeed, N-acetyl-muramyl-L-alanyl D-isoglutamine (1) first synthesized by Merser et al. (49, 50) was found to be fully active (16). Kotani et al. (29, 30) independently came to the same conclusion. 

The proton decoupled nmr spectra of intact cells, isolated cell walls, and cell wall lysozyme digests were obtained for five gram positive bacteria, which have different peptidoglycans and conbinations of accessory polymers, as shown in Figure 2. The chemical shifts of each system is t ulated in Table 1. As seen in Figure 3, each bacteria has a unique intact cell nmr spectrum. The uniqueness of the intact cell spectra reflect differences in the chemical composition and degree of mobility of the cell wall. 

Figure 5.- The gated decoupled CA C E) and broad band noise decoupled CB D/F) nmr spectra of B.subti Lis cells CA B), cell walls CC D), and cell wall Lysozyme digest CE F),...

Figure 6.- The gated decoupled (A C b) and broad band noise decoupled (B,D/F) ISn nmr spectra of H.lysodeikticus cells (A/B) cell walls (B/C)/ and cell wall lysozyme digest.

Figure 8.- The NMR spectra of the Lysozyme digest of B. Lichenifor-mis cell walls stripped of teichoic acid and teichuronic cid (A), the Lysozyme digest of an intact cell wall (B), Lysozyme treated cells in 2 M sucrose (C), and Lysozyme treated cells fixed with glutaraldehyde CD). Spectral conditions as in Figure 1.

The dependence of sample retention (A ) on gradient conditions (/q, A0), flow rate, and column volume is a source of frequent confusion. For example, if we decrease flow rate in the isocratic separation of a protein sample, the sample resolution almost always improves—because the column plate number is increased [Eq. (3)], and values of k and a are not affected. In gradient elution, on the other hand, a decrease in flow rate often leads to a decrease in resolution for some bands and an increase in resolution for others. This is illustrated in Fig. 3 for the separation of a lysozyme digest, where all conditions are the same in these two runs except for flow rate. In Fig. 3a, for a flow rate of 0.5 mL/min, the two bands (arrows) that elute at about 28 min are well resolved. However, with a flow rate of 1 mL/min (Fig. 3b, arrow, 17 min), these two bands merge into a single band. The opposite situation is observed for two bands (arrows) that elute at about 42 min in Fig. 3a and 31 min in Fig. 3b. 

Figure 3 Flow rate effects in gradient elution. Separation of peptide mixture (lysozyme digest) by reversed-phase gradient elution. Gradient time 30 min 8-40% acetonitrile-water (0.1% TFA) gradient Cg column, (a) 0.5mL/min and (b) 1.0 mL/min. (From Ref. 6.)...

Figure 7 Effect of gradient range on reversed-phase separation of lysozyme digest, with Iq/Ai and k held constant. Cg column, 1.9mL/min. (a) 5-60% acetonitrile-water (0.1 % TFA), 30-min gradient, (b) 12-34%, 12 min. (From Ref. 9.)...

The exo-p-D-2-acetamido-3-0-(D-l-carboxyethyl)-2-deoxyglucanase exo- -N-acetylmuramidase) produced by Bacillus subtilis growing under defined conditions is partly secreted and partly bound to the cell wall. The synthesis of this enzyme was induced, in decreasing order of efficiency, by a lysozymal digest of Micrococcus lysodeikticus cell walls, 2-acetamido-4-0-(2-acetamido-2-deoxy-P-D-gIuco-pyranosyl)-3-0-(D-l-carboxyethyl)-2-deoxy-D-glucopyranose, and 2-acetamido-4-0-[2-acetamido- 3-0-(d- 1 -carboxyethyl)-2-deoxy- P-D-glucopyranosyl]-2-deox y-D-... 

Table 5- Muropeptide patterns after lysozyme digestion of cell walls. (% found in muropeptide resp. DAP and lysine per 100% total radioactivity)...

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