Nucleation and growth of microbial lipase crystals from clarified concentrated fermentation broths

Bulk crystallization is emerging as a new industrial operation for protein recovery. Characterization of bulk protein crystallization is more complex than protein crystallization for structural study where single crystals are grown in flow cells. This is because both nucleation and crystal growth processes are taking place while the supersaturation falls. An algorithm is presented to characterize crystallization using the rates of the two kinetic processes, nucleation and growth. The values of these rates allow ready comparison of the crystallization process under different operating conditions. The crystallization, via adjustment to the isoelectric pH of a fungal lipase from clarified fermentation broth, is described for a batch stirred reactor. A maximum nucleation rate of five to six crystals formed per microliter of suspension per second and a high power dependency ( approximately 11) on the degree of supersaturation were found. The suspended protein crystals were found to grow at a rate of up to 15-20 nm/s and also to exhibit a high power dependency ( approximately 6) of growth rate on the degree of supersaturation.     

Protein crystal growth. Growth kinetics for tetragonal lysozyme crystals

A method for immobilizing protein crystals has been devised for determining face growth rates, and used to investigate the growth kinetics of hen egg white lysozyme crystals. Growth rates were determined at 22 degrees C in 0.1 M sodium acetate, 5% NaCl, pH 4.0, on the visually identified (110) face of tetragonal lysozyme crystals. Protein concentrations ranged from 13 to 57 mg/ml (saturation concentration = 1.7 mg/ml). Growth rate data were fit to the equation R = kappa sigma ri, where R = rate in cm/s; kappa = constant; sigma i = solute growth interface supersaturation; and r = rate dependence upon super-saturation, with the result that kappa = 0.146 X 10(-8) cm/s and r = 2.0. A model of the growth process was developed and the experimental data were used to determine the relative roles of transport and interfacial kinetics in the growth of this crystal. Values for the width of the boundary layer delta, the interfacial concentration Ci, and growth rate R were determined. The model may be used to extrapolate to other growth conditions. The relative role of transport and interfacial kinetics can be expressed by the coefficient gamma = (CB - Ci)/(CB - Cs), when CB is the bulk concentration and Cs the saturation. Values for gamma were found to range from much less than 0.1 for submicron-size crystals to approximately 0.15 for cm sizes. The results indicate that attachment or surface effects are rate-limiting in lysozyme crystal growth in Earth's gravity because solutal convection always provides more transport of solute than can be accommodated by the interface. In order to grow such crystals under transport limiting conditions, it would be necessary to suppress this solutal convection.     

Studies of crystal growth mechanisms of proteins by electron microscopy

We have used electron microscopy to examine the surfaces of lysozyme crystals and deduce mechanisms of crystal growth. We find that growth occurs by a lattice defect mechanism at low supersaturation and by two-dimensional nucleation at high supersaturation. Step velocities and two-dimensional nucleation rates are obtained, and their dependence on supersaturation is compared with theory. Some features of the observed surface structure can be related to the specific topology and strengths of the bonds in the P4(3)2(1)2 lattice. Preliminary results on the early stages of nucleation and the phenomenon of cessation of growth are presented.     

Molecular dynamics study of polyethylene chain non-isothermal crystallisation: effects of chain length and branch structure

The molecular dynamics simulations in this work were aimed to provide a molecular insight into chain structure effects on non-isothermal crystallisation of polyethylene (PE) chains. The crystallisation behaviours were influenced by chain length and cooling rate in linear PE chain crystallisation: C100 and C150 were unable to fold into crystals. From C1000 to C3000, crystallisation abilities became stronger as chain length increased. From C5000 to C14000, chain length had no influence on crystallisation abilities. Final morphologies changed from rotator phase to single crystal domain, and to multi crystal domains as chain length increased. The formation of multi crystal domains with longer chain was easier than with the shorter chain in identical conditions. Branch content influenced not only the crystallisation kinetics but also final morphologies in non-isothermal crystallisation. The branches were defective in nucleation process, which was reflected in the crystal growth process. Crystallisation temperature, rate and crystallinity decreased, and the morphologies became disordered as branch content increased. Changes of final morphologies from single crystal domain to multi crystal domains were found under the influence of branch content and cooling rate. Trans-rich phenomenon was observed, and the trans-state population increment was prior to crystallinity increment. Crystallisation processes began at different crystallisation temperature when the trans-state populations reached a critical value which was independent of branch content.     

Comparative differential scanning calorimetric analysis of vegetable oils: II. Effects of cooling rate variation

The effects of scanning rates (1, 5, 10 and 20 degrees C/min) on the DSC cooling profiles of 11 vegetable oils have been determined in order to monitor peak transition temperatures, onset temperatures and crystallisation enthalpies. Triacylglycerol (TAG) profiles and iodine value analyses were used to complement the DSC data. The melted samples exhibited complicated crystallising exotherms. As the cooling rate increased, the crystallisation temperature decreased and the breadth of the crystallisation exotherm on cooling from the melt increased. In addition, the intensity of the exothermic peak increased somewhat when the cooling rate was increased. At slow cooling rates, TAG had more time to interact. It is conceivable that, at a low cooling rate (1 degree C/min), a prominent exotherm would be observed on crystallisation of vegetable oils and fats. The occurrence of one exotherm upon cooling indicated the co-crystallisation of the TAG upon slow cooling. On the basis of the corollary results obtained, vegetable oils may be differentiated by their onset temperature (Ton) values in the DSC cooling curves. Generally, there was a shift of Ton toward lower values with increasing cooling rates.     

The effect of temperature and solution pH on the nucleation of tetragonal lysozyme crystals.

Part of the challenge of macromolecular crystal growth for structure determination is obtaining crystals with a volume suitable for x-ray analysis. In this respect an understanding of the effect of solution conditions on macromolecule nucleation rates is advantageous. This study investigated the effects of supersaturation, temperature, and pH on the nucleation rate of tetragonal lysozyme crystals. Batch crystallization plates were prepared at given solution concentrations and incubated at set temperatures over 1 week. The number of crystals per well with their size and axial ratios were recorded and correlated with solution conditions. Crystal numbers were found to increase with increasing supersaturation and temperature. The most significant variable, however, was pH; crystal numbers changed by two orders of magnitude over the pH range 4.0-5.2. Crystal size also varied with solution conditions, with the largest crystals obtained at pH 5.2. Having optimized the crystallization conditions, we prepared a batch of crystals under the same initial conditions, and 50 of these crystals were analyzed by x-ray diffraction techniques. The results indicate that even under the same crystallization conditions, a marked variation in crystal properties exists.     

Effects of high pressure on the solubility and growth kinetics of monoclinic lysozyme crystals.

Average growth rates of the (0 1 0) and (0 1 0) faces (R<0 1 0>) of monoclinic lysozyme crystals were measured in situ under 0.1 and 100 MPa. From the dependence of the growth rates on the lysozyme concentration, we determined the solubility of the crystal as a function of temperature at 0.1 and 100 MPa. The solubility increased with an increase in pressure. From the comparison between the growth rates under 0.1 and 100 MPa at the same supersaturation level, we found that the growth rates of the monoclinic lysozyme crystals kinetically increase with an increase in pressure. Supersaturation dependencies of the growth rates under 0.1 and 100 MPa were well fitted with a two-dimensional (2D) nucleation growth model of a birth-and-spread type. The fitting results suggest that the increase in the growth rates with pressure can be explained by the decrease in the average ledge surface energy of 2D island, the average distance between the kinks on a step and the activation energies in the incorporation processes of solute molecules.     

A simple preparation method of crystals of soybean hull peroxidase

Soybean hull peroxidase (SHP) was crystallised from an enzyme solution with low purity by a simple method. The enzyme solution was purified by cooperation salting out of acetone and ammonium sulphate, and lumpy crystals were obtained with the size of about 40 × 30 μm when ammonium sulphate was quickly added to the enzyme solution. The crystal was examined and confirmed to be an SHP crystal by the method of activity test. The result shows that, though the purity of the enzyme solution was not high, crystals could be formed when the enzyme solution rapidly reached to a degree of supersaturation, which was different from the traditional methods of protein crystallisation. Additionally, a purification method of acetone and ammonium sulphate fractional salting out was also studied, in which the procedure was simplified, and a satisfactory purification effect was obtained.     

Growth of Ca-D-malate crystals in a bioreactor

To develop a bioreactor for solid-to-solid conversions, the conversion of solid Ca-maleate to solid Ca-D-malate by permeabilized Pseudomonas pseudoalcaligenes was studied. In a bioreactor seeded with product (Ca-D-malate) crystals, growth of Ca-D-malate crystals is the last step in the solid-to-solid conversion and is described here. Crystal growth is described as a transport process followed by surface processes. In contrast to the linear rate law obeyed by the transport process, the surface processes of a crystal-growth process can also obey a parabolic or exponential rate law. Growth of Ca-D-malate crystals from a supersaturated aqueous solution was found to be surface-controlled and obeyed an exponential rate law. Based on this rate law, a kinetic model was developed which describes the decrease in supersaturation due to Ca-D-malate crystal growth as a function of the constituent ions, Ca(2+) and D-malate(2-). The kinetic parameters depended on temperature, but, as expected (surface-controlled), they were hardly affected by the stirring speed.     

Physical properties of poly(hydroxybutyrate) and copolymers of hydroxybutyrate and hydroxyvalerate

Abstract The major physical properties of poly(hydroxybutyrate) (PHB) and its copolymers with hydroxyvalerate (PHB/HV) and those of their blends are summarised. Cases of liquid-liquid phase separation in homopolymer copolymer blends are reported and conditions where further phase separation occurs during crystallisation are documented. The crystallinity and degree of comonomer inclusion in the crystallisation of copolymers are described and quantified. It is argued that the mechanism for the inclusion of comonomer units in the crystals is of a kinetic rather than an equilibrium nature.     

Probabilistic approach to lysozyme crystal nucleation kinetics

Nucleation of lysozyme crystals in quiescent solutions at a regime of progressive nucleation is investigated under an optical microscope at conditions of constant supersaturation. A method based on the stochastic nature of crystal nucleation and using discrete time sampling of small solution volumes for the presence or absence of detectable crystals is developed. It allows probabilities for crystal detection to be experimentally estimated. One hundred single samplings were used for each probability determination for 18 time intervals and six lysozyme concentrations. Fitting of a particular probability function to experimentally obtained data made possible the direct evaluation of stationary rates for lysozyme crystal nucleation, the time for growth of supernuclei to a detectable size and probability distribution of nucleation times. Obtained stationary nucleation rates were then used for the calculation of other nucleation parameters, such as the kinetic nucleation factor, nucleus size, work for nucleus formation and effective specific surface energy of the nucleus. The experimental method itself is simple and adaptable and can be used for crystal nucleation studies of arbitrary soluble substances with known solubility at particular solution conditions.     

Turning protein crystallisation from an art into a science

Protein crystallisation has gained a new strategic and commercial relevance in the post-genomic era because of its pivotal role in structural genomics. Producing high-quality crystals has always been a bottleneck to structure determination and, with the advent of proteomics, this problem is becoming increasingly acute. The task of producing suitable crystals may be tackled using two approaches. The first relies on empirical techniques that are based mainly on trial and error, and what is perceived to be the 'art' of crystallisation. The second approach is aimed at gaining an understanding of the fundamental principles that govern crystallisation; this knowledge may be applied to design experimental methodology for producing high-quality crystals of medical and industrial interest.     

Cholesterol crystal formation and growth in model bile solutions

Cholesterol monohydrate crystal formation was studied in supersaturated model bile solutions, containing unlabeled cholesterol, sodium cholate and soybean phosphatidylcholine, and tracer amounts of [3H]cholesterol. Solutions were either seeded with cholesterol crystals to initiate growth, or not seeded to allow self-nucleation and subsequent crystal growth to occur. Crystal growth at 37 degrees C was measured by two methods. First, radioactive cholesterol crystals were isolated by filtration, and the mass of cholesterol that had precipitated was calculated. In unseeded solutions, there was a long lag period before crystal growth was detected. This lag time was decreased by increases in the cholesterol concentration, temperature, and lipid concentration. In seeded solutions, crystal growth also was dependent on the cholesterol concentration, temperature, and lipid concentration. The second method used to measure crystal growth involved the Coulter Counter. At 37 degrees C, reproducible results were not obtained using unseeded solutions due to blocking of the counter aperture with large crystals. In seeded solutions, crystal growth could be measured as an increase in total particle volume. However, comparison of growth rate estimates from the Coulter Counter with those obtained radiochemically revealed poor agreement between the two methods. It is probable that the Coulter Counter is inaccurate in measuring the volume of cholesterol monohydrate crystals due to their anisometric shape.     

Repair of impurity-poisoned protein crystal surfaces

The surface morphology of Bence-Jones protein (BJP) crystals was investigated during growth and dissolution by using in situ atomic force microscopy (AFM). It was shown that over a wide supersaturation range, impurities adsorb on the crystalline surface and ultimately form an impurity adsorption layer that prevents further growth of the crystal. At low undersaturations, this impurity adsorption layer prevents dissolution. At greater undersaturation, dissolution takes place around large particles incorporated into the crystal, leading to etch pits with impurity-free bottoms. On restoration of supersaturation conditions, two-dimensional nucleation takes place on the impurity-free bottoms of these etch pits. After new growth layers fill in the etch pits, they cover the impurity-poisoned top layer of the crystal face. This leads to the resumption of its growth. Formation of an impurity-adsorption layer can explain the termination of growth of macromolecular crystals that has been widely noted. Growth-dissolution-growth cycles could be used to produce larger crystals that otherwise would have stopped growing because of impurity poisoning.     

Orientated growth of crystalline anhydrous maltitol (4-O-alpha-D-glucopyranosyl-D-glucitol)

In the industrial crystallisation process of maltitol (C(12)H(24)O(11)), the presence of maltotriitol (C(18)H(34)O(16)) in the maltose syrup is responsible for a change of the crystal morphology. Two different crystal forms of maltitol were obtained: a prismatic one and a 'bipyramidal' one. IR and X-ray diffraction experiments (single crystal and powder) were performed to identify both crystal parameters. It is concluded that a structural polymorphism has to be ruled out. Close coincidences in some of the crystal cell parameters of maltitol and maltotriitol allow to assume the process through which maltotriitol affects the morphology of maltitol crystals.     

Differences in growth and water relations among Phaseolus vulgaris cultivars in response to induced drought stress

Relatively little ecophysiological research has been conducted to determine the responses to drought of Phaseolus vulgaris. Four bean cultivars (cvs.) from Brazil, A320, Carioca, Ouro Negro and Xodó were submitted to an imposed water deficit in order to evaluate the importance of some adaptive mechanisms of drought resistance through the analysis of growth parameters, water status, gas exchange and indicators of tolerance mechanisms at the cellular level. During the drought treatment, relative growth rates were more reduced for A320 and Xodó than Carioca and Ouro Negro. A320 closed its stomata very rapidly and complete stomatal closure was obtained at Psi(w)=-0.6 MPa, in contrast to the other cvs. where stomata were fully closed only at Psi(w)=-0.9 MPa. Net assimilation rates were closely related to stomatal conductances. Mechanisms at the cellular level appeared to be mostly important for higher tolerance. Carioca and Ouro Negro, when compared to A320 and Xodó, were characterized by having better drought tolerance mechanisms and higher tissue water retention capacity leading to a better growth under water deficits. The leaf dehydration rates of those cvs. were slow whereas those of the drought sensitive cvs. were rapid. The results were confirmed by the electrolyte leakage test and leaf osmotic potential measurements, which indicated higher membrane resistance and osmotic adjustment in the two tolerant cvs. Carioca and Ouro Negro. It appears from this study that despite being cultivated in the same geographical region, the four cvs. of P. vulgaris displayed somewhat different drought adaptive capacities for prolonged drought during the vegetative phase.     

Protein purification by bulk crystallization: the recovery of ovalbumin

Crystallization is used industrially for the recovery and purification of many inorganic and organic materials. However, very little is reported on the application of bulk crystallization for proteins. In this work, ovalbumin was selected as a model protein to investigate the feasibility of using bulk crystallization for the recovery and purification of proteins. A stirred 1-L seeded batch crystallizer was used to obtain the crystal growth kinetics of ovalbumin in ammonium sulfate solutions at 30 degrees C. The width of the metastable region, in which crystal growth can occur without any nucleation, is equivalent to a relative supersaturation of about 20. The bulk crystallizations were undertaken within this range (using initial relative supersaturations less than 10) and nucleation was not observed. The ovalbumin concentration in solution was measured by UV absorbance and checked by crystal content measurement. Crystal size distributions were measured both by using a Malvern Mastersizer and by counting crystals through a microscope. The crystal growth rate was found to have a second-order dependence upon the ovalbumin supersaturation. While there is no discernible effect of ammonium sulfate concentration at pH 4.90, there is a slight effect at higher pH values. Overall the effect of ammonium sulfate concentration is small compared to the effect of pH, for which there is a 10-fold increase in the growth rate constant, k(Gsigma) over the range pH 4.6-5.4. To demonstrate the degree of purification which can be achieved by bulk crystallization, ovalbumin was crystallized from a solution containing conalbumin (80,000 Da) and lysozyme (14, 600 Da). After one crystallization and a crystal wash, ovalbumin crystals were produced with a protein purity greater than 99%. No contamination by the other proteins was observed when using overloaded sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) stained with Coomassie blue stain and only trace amounts of lysozyme were observed using a silver stain. The presence of these other proteins in solution did not effect the crystal growth rate constant, k(Gsigma). The study demonstrates the feasibility of using bulk crystallization for the recovery and purification of ovalbumin. It should be readily applicable to other protein systems. (c) 1995 John Wiley & Sons, Inc.     

Visualization of RNA crystal growth by atomic force microscopy.

The crystallization of transfer RNA (tRNA) was investigated using atomic force microscopy (AFM) over the temperature range from 4 to 16 degrees C, and this produced the first in situ AFM images of developing nucleic acid crystals. The growth of the (110) face of hexagonal yeast tRNAPhe crystals was observed to occur at steps on vicinal hillocks generated by multiple screw dislocation sources in the temperature range of 13.5-16 degrees C. Two-dimensional nucleation begins to dominate at 13.5 degrees C, with the appearance of three-dimensional nuclei at 12 degrees C. The changes in growth mechanisms are correlated with variations in supersaturation which is higher in the low temperature range. Growth of tRNA crystals was characterized by a strong anisotropy in the tangential step movement and transformation of growth modes on single crystals were directly observed by AFM over the narrow temperature range utilized. Finally, lattice resolution images of the molecular structure of surface layers were recorded. The implications of the strong temperature dependence of tRNAPhe crystal growth are discussed in view of improving and better controlling crystallization of nucleic acids.     

Cryopreservation of canine spermatozoa: theoretical prediction of optimal cooling rates in the presence and absence of cryoprotective agents

In the present study a shape independent differential scanning calorimeter (DSC) technique was used to measure the dehydration response during freezing of ejaculated canine sperm cells. Volumetric shrinkage during freezing of canine sperm cell suspensions was obtained at cooling rates of 5 and 10 degrees C/min in the presence of extracellular ice and CPAs (6 different combinations of freezing media were used, ranging from a media with no CPAs, and those with 0.5%, 3%, and 6% glycerol and with 0.5% and 3% Me(2)SO). Using previously published data, the canine sperm cell was modeled as a cylinder of length 105.7mum and a radius of 0.32mum with an osmotically inactive cell volume, V(b), of 0.6 V(o), where V(o) is the isotonic cell volume. By fitting a model of water transport to the experimentally obtained volumetric shrinkage data the best fit membrane permeability parameters (L(pg) and E(Lp)) were determined. The "combined best fit" membrane permeability parameters at 5 and 10 degrees C/min for canine sperm cells in the absence of CPAs are: L(pg)=0.52x10(-15)m(3)/Ns (0.0029mum/min-atm) and E(Lp)=64.0kJ/mol (15.3kcal/mol) (R(2)=0.99); and the corresponding parameters in the presence of CPAs ranged from L(pg)[cpa]=0.46 to 0.53x10(-15) m(3)/Ns (0.0027-0.0031mum/min-atm) and E(Lp)[cpa]=46.4-56.0kJ/mol (11.1-13.4kcal/mol). These parameters are significantly different than previously published parameters for canine and other mammalian sperm obtained at suprazero temperatures and at subzero temperatures in the absence of extracellular ice. The parameters obtained in this study also suggest that optimal rates of freezing canine sperm cells ranges from 10 to 30 degrees C/min; these theoretical cooling rates are found to be in close conformity with previously published but empirically determined optimal cooling rates.     

Protein nano-crystallogenesis

We demonstrate the feasibility of growing crystals of protein in volumes as small as 1 nanoliter. Advances in the handling of very small volumes (i.e. through inkjet and other technologies) open the way towards fully automated systems. The rationale for these experiments is the desire to develop a system that speeds up the structure determination of proteins by crystallographic techniques, where most of the precious protein sample is wasted for the identification of the ideal crystallisation conditions. An additional potential benefit of crystallisation in very small volumes is the potential improvement of the crystal quality through reduced convection during crystal growth. Furthermore, in such small volumes even very highly supersaturated conditions can be stable for prolonged periods, allowing additional regions of phase-space to be prospected for elusive crystallisation conditions. A massive improvement in the efficiency of protein crystallogenesis will cause a paradigm shift in the biomolecular sciences and will have a major impact in product development in (for example) the pharmaceutical industry.