biomaterial crystallization. bioprocess technology

1. (CRYSTALLIZATION AND STABILITY OF DIFFERENT
PROTEIN CRYSTAL MODIFICATIONS : A CASE STUDY
OF LYSOZYME)
SUBMITTED BY :
SAWAN BHAT
CB21M15

2. INTRODUCTION
• Crystallization from solution is a separation technique where a
solid phase is separated from a mother liquor. The process
consists of two major steps, nucleation and crystal growth.
• Nucleation is the step where the solute molecules dispersed in
the solvent start to gather into clusters.
• Crystal growth is the rate of displacement of a given crystal
surface In the direction perpendicular to the face.
• In this case study we will see, the crystallization, including both
the phase diagram and the phase transition of hen egg white
lysozyme (HEWL)

3. • Egg white lysozyme of chicken eggs belongs to the c-type
lysozymes ,which are one of the major types identified in the
animal kingdom, along with g-type (goose-type and bacterial
lysozyme) and i-type (invertebrate lysozyme).
• Lysozyme is endowed with a number of properties suitable for
food application. It is a heat-stable protein and is not
inactivated by solvents and does not lose its activity when
redissolved in water.
• Applications of Lysozyme include , cheese preservation ,
preservative in either pasteurized or sterile filtered beer, used
in extension of shelf life of various food items such as
vegetables.

4. CASE STUDY
• A tetragonal modification and a needle modification were
obtained during crystallization.
• The phase diagram and stability of two modifications in both
acid and basic pH solutions (pH 4.5, 8.0 and 9.0) were
obtained.
• Based on the phase diagram, phase transfer behavior was
found to exist between the two modifications.
• Change of temperature and pH were studied on the crystal
structure.

5. • A model system for investigations into protein crystallization is
the enzyme hen egg white lysozyme (HEWL). This protein can
crystallize in different crystal forms by using different
precipitants and by varying crystallization conditions like
temperature, pH, supersaturation level and with the presence of
small amounts of heavy atoms and traces of other materials.
• Different morphologies that have been discovered till now ,
some of them are, using sodium chloride as the precipitant, the
well-known tetragonal structure P43212 can be obtained from a
sodium acetate buffer in a temperature ranging from −4 to 25
°C.
• An orthorhombic structure P212121 (HTO) crystallizes from the
acid buffer solution between 25 and 60 °C. This kind of HTO

6. • In addition, studies have also found that phase transformation can
happen between different modifications of protein crystals. At
temperatures higher than 25 °C, the tetragonal morphology will
transfer to the orthorhombic modification (HTO- high temperature
orthorhombic) with the existence of acid buffer solutions.
• This case study is concerned with investigations of both phase
diagram and the phase transition of different protein modifications.
• Hen egg white lysozyme (HEWL) was chosen as the model protein.
Tetragonal morphology and needle shaped crystals were obtained
from the crystallization progress. Both kinds of crystals were
identified by morphology and X-ray powder diffraction (XRPD)
determination.
• Phase diagrams were made in both acid and basic mediums and

7. EXPERIMENTAL
• Lysozyme crystallization :
Hen egg white lysozyme (HEWL) was purchased from Fluka , and
used without further purification.
Sodium chloride (NaCl) purchased from Carl Roth was chosen as the
precipitant. All the protein and salt solutions were treated with buffer
solutions.
Lysozyme was dissolved in buffer and the precipitate solution was
prepared by dissolving sodium chloride in the same buffer.
The tetragonal crystals were prepared from a solution containing 8
mg/mL protein and 4% (w/v) NaCl.
The crystallization process of the tetragonal modification was carried
out in both 0.1 M sodium acetate buffer (pH 4.5) and 0.5 M Tris-HCl
buffer (pH 8.5).

8. The needle shaped crystals were prepared from a solution with
a final concentration of 12 mg/mL protein and 4% (w/v) NaCl in
0.5 M Tris-HCl buffer (pH 8.5).
All the crystallization solutions were stored at 7 °C for
crystallization.
When the crystallization process finished, 100 μL of the
crystallization solution was taken out for the microscope
analysis. The mother liquor and crystals were separated by
centrifugation.

9. X-RAY POWDER DIFFRACTION (XRPD)
• X-ray powder diffraction was carried out at room temperature
on a Brucker D8 Discover diffractometer equipped with a Cu Kα
source.
• X-ray diffraction analysis (XRD) is a technique used in materials
science to determine the crystallographic structure of a
material
• The measurement is used to compare the crystal structure of
different crystal forms.
• Diffraction data were collected under 40 kV, 40 mA. Typical

10. ACTIVITY TEST
• The activity test was carried out by the use of a Varian Cary 50
UV/Vis spectrophotometer.
• The bacteria, M. lysodeikticus, of 8.4 mg were suspended in 50
mL 66mM phosphate buffer, pH 6.2 and used as the substrate.
• The crystalline enzyme solutions (1 mg/mL) of tetragonal and
needle forms were prepared with the same buffer.
• The biological reaction was carried out by adding 100 μL
enzyme solution to 2.5 mL bacteria solution at 25 °C.
• The decreasing in the concentration of the bacteria in 2 min
was monitored by a photometer at the wavelength of 450 nm.

11. SOLUBILITY MEASUREMENT
• The solubility measurements were carried out in a STEM
Integrity Parallel Synthesis Station.
• To determine the solubility points, lysozyme crystal
suspensions were heated up to a certain temperature. A
heating rate of 0.1 K/min was involved here. The solubility
point is defined as the point when transmission reaches a
stable plateau.
• The solubility of different lysozyme modifications was
measured at three different pH values (pH = 4.5, 8.0 and 9.0).
• The concentration of NaCl remained 4% (w/v) for all
measurements. The protein concentration of the clear solution
was verified by UV/Vis spectrophotometer at a wavelength of
280 nm after each experiment

12. PHASE TRANSITION EXPERIMENT
• The phase transition between different modifications was
observed by a microscope.
• A cell was filled with 2 mL of protein solution by a pipette, and
seed crystals were added to the solution.
• The temperature of the cell was controlled by a thermostat.
• Phase transition between tetragonal and needle modifications
were observed under different pH and different temperature.
• The progress was recorded by a microscope equipped with a
digital camera.

13. RESULTS AND DISCUSSION
• Lysozyme crystallization and crystal morphology :
(a) Tetragonal crystals from 0.1M sodium acetate buffer pH 4.5
(8 mg/mL)
(b) Tetragonal crystals from 0.5M Tris-HCl buffer pH 8.5 (8
mg/mL).
(c) Needle shaped crystals from 0.5M Tris-HCl buffer pH 8.5
(12 mg/mL).

14. XPRD MEASUREMENT
• The XRPD pattern of tetragonal and needle forms obtained from the
experiment are shown in fig. below.
• Two intense peaks in the needle pattern, at about 5 degree and 7
degree, are missing in the tetragonal pattern.
• The pattern for the buffer and salt were also measured and the
results were quite different from protein crystals, since the peaks of
buffer and salt appear at higher degrees than most of the peaks for
protein crystals.
• It can be a proof that the two lysozyme morphologies have different
crystal structures

15. SOLUBILITY AND PHASE DIAGRAM
• The solubility was checked by the turbidity measurement and the method
has been successfully used for measuring the solubility of protein crystals.
• By measuring the solubility of different morphologies of lysozyme, it is
possible to determine the stability of the different crystal forms.
• The solubility of both forms increases with the temperature as normal
behavior. However, the effect of pH is not as clear as that of temperature.
• The solubility of needle shaped crystals decreases with an increase of the
pH. But the relationship between solubility and pH for tetragonal crystals is
not that obvious.

16. PHASE TRANSITION
• Phase transformations of different lysozyme morphologies have been observed in a microscope cell.
• It was found in the literature, the transition temperature from tetragonal to HTO lysozyme crystals
was around 25 °C and LTO modification can transfer to HTO form when the temperature is higher than
35 °C.
• The phase transition from needle shape too tetragonal morphology was carried out in acid conditions.
• For the transformation experiment, a lysozyme solution with a concentration of 5 mg/mL was
prepared in 0.1 M sodium acetate buffer.
• Seed crystals of the needle morphology, without special treatment, were added to the solution. The
solution was kept at 20 °C to avoid transformation to the HTO modification and no change in the pH,
ionic strength and protein concentration were involved.
Fig. Phase transformation of needle shaped to tetragonal crystals. The process was carried
out in 0.1 M sodium acetated buffer at 20 °C

17. • The phase transition process can be more complicated in basic
buffer solutions. When pH increases to 9, needle shaped
crystals are more stable according to the phase diagram.
• The phase transition process in pH 9 buffer solutions was
observed in a microscope cell.
• Tetragonal seed crystals were added to a protein solution of 8
mg/mL in 0.5 M Tris-HCl buffer at 20 °C.

18. • The effect of pH was studied on these sample. A lysozyme
solution of 8 mg/mL was prepared with pH 8, pH 8.5 and pH 9
buffer, respectively and all the experiments were carried out at
20 °C.
Fig. Effect of pH on the phase transition behavior. All
experiments were carried out at 20 °C. A: pH 9, B: pH 8.5, and C:
pH 8.

19. • Experiments found that tetragonal crystals can still be obtained
at low temperature with the existence of needle shaped
crystals.
Effect of temperature on the phase transition behavior. A: 35 °C,
B: 30 °C, C: room temperature, D: 7 °C.

20. CONCLUSION
• The phase diagram and phase transition of hen egg white lysozyme
were studied.
• Tetragonal and needle modifications were investigated and the stable
regions of both modifications were determined.
• It is more likely to obtain the tetragonal crystal form in an acid
solution, and in solutions with a pH 9, needle shaped crystal forms
nucleate more likely.
• The tetragonal modification can be obtained in a basic solution as
well, but is only stable at low temperature.
• In addition to the transition behaviors already discovered, phase
transformation between tetragonal modification and needle shaped
crystals was found.
• In acid buffer solutions, the needle shaped crystals can, transfer to