Lysozyme procedure

Figures 13 and 14 respectively show the relationship of average trans membrane pressure and circulation rate on flux for both whole cells and lysate suspensions. The lysate was produced via the lysozyme procedure. Qualitatively, the behaviors are quite similar to those seen in Figures 9 and 10 where the cells are lysed by sonication. Beyond minimum circulation flows, little extra flux is attained as the circulation rate increases. The average TMP is the dominant factor in producing flux, however, if Pin - Pout less than about 20 psi, debris can accumulate at the membrane surface and inhibit flow. It should be emphasized that these results pertain to 0.45 micron pore size microporous membrane only.

Figure 13. Influence of transmembrane pressure on flux (0.45 /im microporous). Lysate was produced via the lysozyme procedure. The same general behavior as for sonified lysate is seen here.

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. 

In one case, a small peptide with enzyme-like capability has been claimed. On the basis of model building and conformation studies, the peptide Glu-Phe-Ala-Ala-Glu-Glu-Phe-Ala-Ser-Phe was synthesized in the hope that the carboxyl groups in the center of the model would act like the carboxyl groups in lysozyme 17). The kinetic data in this article come from assays of cell wall lysis of M. lysodeikticus, chitin hydrolysis, and dextran hydrolysis. All of these assays are turbidimetric. Although details of the assay procedures were not given, the final equilibrium positions are apparently different for the reaction catalyzed by lysozyme and the reaction catalyzed by the decapeptide. Similar peptide models for proteases were made on the basis of empirical rules for predicting polypeptide conformations. These materials had no amidase activity and esterase activity only slightly better than that of histidine 59, 60). 

In an earlier experiment, Jori et al. (14) reported that methionyl residues are important in maintaining the tertiary structure of lysozyme. The introduction of a polar center into the aliphatic side chain of methionine, as a consequence of the conversion of the thioether function to the sulfoxide, may bring about a structural change of the lysozyme molecule which, in turn, reduces the catalytic efficiency. When ozonized lysozyme was treated with 2-mercaptoethanol in an aqueous solution according to the procedure of Jori e al. (14), the enzyme did not show any increase in its activity. This may be explained in two ways. In one, such reactions are complicated by many side reactions, e.g. sulfhydryl-disulfide interchange, aggregation and precipitation of the modified enzyme (24-26). In the other, the failure to recover the activity of the enzyme may by associated with the extensive oxidation of other residues. 

C2H5/heptane/ buffer AOT/isooctane Ribonuclease A, cytochrome C, lysozyme Fisher s polarity ratio (P) linearly related to hydrophilic surroimdings at minimal AOT concentration for 100 % forward extraction. A procedure to estimate the AOT levels required for protein extraction was proposed [72]... 

The preferred modem procedure for investigating an enzyme mechanism is to identify a protein in a genome sequence, clone and express it, solve the crystal and/or solution structures, guess the mechanism, and then confirm it by site-directed mutagenesis and kinetics. The mechanism of lysozyme almost illustrates that procedure. Although the classical protein chemistry on lysozyme predated the molecular genetics, the kinetic studies were stimulated by the x-ray crystallography, and the previously unknown mechanism almost leapt from the... 

Figure 3.18 Schematic diagram of the hen egg lysozyme refolding procedure with the help of ethane-bridged PMOs.241 (Reprinted with permission from X. Wang et al., Langmuir 2007, 23, 5735-5739. Copyright 2007 American Chemical Society.) (See color insert.)...

The procedures and calculations described in this chapter provide considerable insight into the factors affecting the conformations of polypeptides and proteins. The computer programs for gramicidin-S, oxytocin, vasopressin, etc., can also be used for larger structures—of the size of ribonuclease and lysozyme—although the required computer time is considerably increased. 

The conjugation of lysozyme and carboxymethyl morphine is shown schematically in Figure 15. In this procedure, about four morphines are attached to the enzyme. 

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