Lysozyme monitoring

Kunichika, K., Hashimoto, Y. and Imoto, T. (2002) Robustness of hen lysozyme monitored by random mutations. Protein Engineering, 15, 805-809. 

Schwartz, A. M., and Berglund, K. 2000. In situ monitoring and control of lysozyme concentration during crystallization in a hanging drop. J. Cryst. Growth 219 753-60. 

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). 

Schwartz et al. evaluated the use of fibre-optic probe dispersive Raman and PLS calibration to study crystallisation in a hanging drop experiment. They could show that the lysosome concentration could be monitored during the experiment through the concentration phase, nucleation and crystal growth [27], In later experiments, they were able to utilise in-line Raman data to control the concentration of lysozyme to affect the crystallisation path and thereby obtaining the desired large single crystals [28], The same setup was used for crystallisation of apoprotein. Also in this case the Raman data were used to control the evaporation and thus the supersaturation for optimised crystal size [29]. 

The assay principle is straightforward the clearance of a Micrococcus hocus suspension caused by lysozyme is monitored at 450 nm and compared to a lysozyme standard of known activity. Expression of lysozyme activity in absolute terms is dependent on the substrate sensitivity. Moreover (he light scattering effect of the suspension is determined by the optical geometry of (he spectrophotometer. Therefore, absolute turbidity values can only be obtained by calibration of the apparatus with a standard of known turbidity. 

Activity is measured by the procedure of Shugar.21 To 2.9-ml cuvettes (1 cm path length), diluted lysozyme (ranging from 0.1 to 0.5 nM) and antibody (ranging from 0.013 to 50 nM) are added to 66 mM potassium phosphate buffer, pH 6.24, and 0.1% bovine serum albumin (BSA) (w/v) to a volume of 900 fil. The solutions are kept at 25° for 1 hr to allow the lysozyme-antibody complexes to come to equilibrium. The activity assays are initiated by the addition of 100 pi Micrococcus lysodeikticus (Sigma Chemical Company) cell walls (2 mg/ml in 66 mM potassium phosphate, pH 6.24) to a final A450 of 0.8 -1.0. Cuvettes are wrapped with Parafilm to prevent evaporation, inverted several times to mix, and placed in a Perkin-Elmer, Norwalk, CT) Lambda 4B spectrophotometer. Reactions are monitored by the decrease in A450 for 70 min with a data point collected every minute. 

The culture is harvested by centrifugation at 5000 g and 4° at peak production. Lysozyme activity, which is monitored by the standard turbidity assay of culture supernatant (described in a later section), typically reaches its peak after 5 to 7 days of growth in rich medium. For inactive mutants, the peak of lysozyme secretion is estimated as 12 to 24 hr after the yeast cell density (monitored by A6S0) reaches its peak. 

The transition midpoints (Tm) of lysozymes can be monitored directly with a spectrophotometer equipped with an automatic temperature-stepping module (e.g., the Gilford UV-Vis apparatus, Oberlin, OH). The decrease in absorbance at 292 nm with rising temperature reflects the shift of the internal tryptophan residues to an aqueous environment,31 and hence... 

Fig. 3. The transition midpoint (rn) of thermal denaturation of a synthetic lysozyme is determined spectrophotometrically by monitoring the decrease in the absorbance at 292 nm with rising temperature. The thermally induced transition for CL is cooperative and obeys the two-state model to a good approximation.52...

Fig. 4. Profile of a differential scanning calorimetry experiment done on a synthetic lysozyme. The heat capacity (kilocalories per degree per mole) of the unfolding process was monitored as a function of temperature on a Micro-Cal MC2 instrument. The transition midpoint of protein unfolding corresponds to the temperature at the peak of the curve, and the thermodynamic parameters A H and A Cp are evaluated by the procedure of Privalov.33...

FIGURE 8 Displacement histogram and UV detector trace for a selective displacement process. (A) Displacement separation of a three-component protein mixture using streptomycin sulfate A as a displacer. Column 100 X 5 mm i.d. strong cation exchange (8 m) carrier 30 mM sodium phosphate buffer, pH 6.0 feed 1.6 mL of 0.392 mAI ribonudease A, 0.42 mM horse cytochrome c and 0.34 mM lysozyme in the carrier. Total column loading 12.7 mg/mL column displacer 25 mM streptomycin sulfate A flow rate 0.2 mL/min fraction size 200 /iL. (Kundu et al.43) (B) UV detector trace monitored at 280 nm for the displacement separation shown below. 

The protein properties include (1) motions of several proteins monitored by ESR spin labels (Belonogova et al., 1978, 1979 Likhtenshtein, 1976 Steinhoff et al., 1989) and Mossbauer labels (Belonogova et al., 1979 Likhtenshtein, 1976) (2) temperature dependence of neutron scattering for myoglobin (Cusack, 1989 Doster et al., 1989) (3) Mossbauer spectra (Parak et al., 1988) and RSMR spectra (Goldanskii and Krupyanskii, 1989) of myoglobin and (4) mechanical properties of lysozyme crystals (Morozov and Gevorkyan, 1985 Morozov et al., 1988). 

There are several problems requiring careful attention. Lysozyme has a tendency to form complexes with many substances [e.g., alkyl sulfates, fatty acids, aliphatic alcohols (Smith and Stocker, 1949), cephalins (Brusca and Patrono, 1960), and other proteins]. Of particular importance is its tendency to form complexes with transferrins [e.g., ovotrans-ferrin (Ehrenpreis and Warner, 1956)]. These interactions lead to difficulties in the isolation of lysozyme. Some recent workers have used fast protein liquid chromatography (FPLC) and high-performance liquid chromatography (HPLC) (e.g., Ekstrand and Bjorck, 1986). The resolution in these procedures may not always be satisfactory, and in HPLC pressure and solvent effects must be monitored carefully if the product is to be suitable for conformation and activity studies. 

---