Crystallization as a Purification Method for Jack Bean Urease: On the Suitability of Poly(Ethylene Glycol), Li2SO4, and NaCl as Precipitants Maxim Weber, Matthew J. Jones,* and Joachim Ulrich
CRYSTAL GROWTH & DESIGN 2008 VOL. 8, NO. 2 711–716
Martin-Luther-UniVersität Halle-Wittenberg, Zentrum für Ingenieurwissenschaften, Verfahrenstechnik/ TVT, 06099 Halle (Saale), Germany ReceiVed January 23, 2007; ReVised Manuscript ReceiVed October 10, 2007
ABSTRACT: The purification of urease from jack bean meal by means of extraction and crystallization in the presence of different precipitants has been investigated. Purification with poly(ethylene glycol) proves to be a promising alternative to established methods. Although lower yields and purities were observed when compared to Sumner’s method, a suitable combination of process parameter changes has the potential to match or even exceed the product qualities previously obtained. Moreover, poly(ethylene glycol) shows promise as a washing liquid for the final crystal product. In contrast to extraction and crystallization of urease in the presence of acetone, the additive 2-mercaptoethanol has a detrimental effect upon product quality in the case presented here. The salts lithium sulfate and sodium chloride show no effective selective precipitation or crystallization of jack bean proteins.
1. Introduction The rapidly growing demand for proteins, especially enzymes, in different industry branches including the pharmaceutical, chemical, and biochemical sector as well as the food industry merits the investigation of cheap and effective protein purification methods. Frequently, upstream processing of high value proteins with low natural abundance makes use of recombinant microorganisms capable of producing considerably higher concentrations of the desired protein than those available in renewable resources. Perhaps the most well-known example is recombinant insulin.1 Generally the protein cannot be used without further purification, especially in medical applications. Even if the impure protein satisfies quality requirements (as may be the case in technical applications), purification can result in better reproducible properties leading to better control over the final product and reduced costs associated with logistics, in particular when the content of active enzyme is increased.2 Crystallization is a very powerful method for the purification of materials that are solids under ambient conditions. Only few proteins exist for which direct crystallization procedures for the purpose of purification have been reported, among these hen egg white lysozyme, ovalbumin, jack bean urease, and glucose isomerase.3–6 In all cases a final product with high purity is obtained. Chromatographic purification methods are likely to remain indispensable for the foreseeable future where high quality demands have to be met. However, for technical enzymes crystallization is an economically attractive and effective alternative. Replacing chromatographic purification steps by protein crystallization can lead to a dramatic reduction in production costs, as shown by Visuri.6 The reasons for the absence of widespread use of crystallization for the purification of proteins can be found in the paucity of information on the fundamental properties of proteins in general with respect to their crystallization. With the exception of a small number of model proteins such as hen egg white lysozyme, ovalbumin, glucose isomerase, and R-amylase,7–13 to name but a few, little is known about the solubility and * To whom correspondence should be sent. Telephone: +49 345 5528403. Fax: +49 345 5527358. E-mail:
[email protected].
crystallization kinetics of proteins. Complications also arise due to the nature of the raw materials. Apart from proteins they contain a significant number of other substances with varying concentrations. In addition, the amount of different proteins and their individual concentration can vary depending on protein type and provenance of the raw material. Concentrations of ovalbumin in hen egg white can reach 65–70%,14 but numerous proteins, especially enzymes, have low abundance in their source materials. Here, the extraction and crystallization of urease from jack beans (CanaValia ensiformis) in the presence of different precipitants was investigated and compared to the established method of Sumner.15 In his work, Sumner used a 32% (v/v) acetone–water solution to extract jack bean proteins from the meal. The extraction was allowed to proceed for 3–4 min at 28 °C. Thereafter, the extract was placed in an ice chest at 2–2.5 °C overnight. This leads to the crystallization of urease, while the majority of the other proteins remains in the extract. This method followed by recrystallization provides a 700- to 1400-fold increase in urease purity compared to the urease content in the jack beans, with the variance of the effectiveness due to the provenance of the source material.16 This simple and effective method is used to this day with little modification (see, for example, Takishima et al.,17 Fishbein et al.18). However, because of the small yield of the method (ca. 15–20% of the total urease content of the meal, as shown in Weber et al.19) and the detrimental environmental impact of acetone, for large scale production a better method is required. The precipitants employed were poly(ethylene glycol) with mean molar masses of 4000 and 6000 g/mol and the salts lithium sulfate and sodium chloride. The salts were selected on the basis that they represent different extremes in the Hofmeister series.20 Urease has a low abundance in the raw material (jack bean meal, JBM). The total protein content in the meal is ca. 22% of which less than 0.1% is urease. Urease is a large hexamer consisting of six identical units with a molar mass of 91 kDa.21,22 The isoelectric point of urease is at pH ) 5.0–5.1, and it is here that the lowest solubility is expected. Crystals of urease have cubic symmetry with octahedral crystal habit.23,24 To our knowledge, the crystal structure of jack bean urease has not yet been solved to high resolution, although the lattice param-
10.1021/cg070070i CCC: $40.75 2008 American Chemical Society Published on Web 01/05/2008
712 Crystal Growth & Design, Vol. 8, No. 2, 2008
eters and space group symmetry are known. The crystal size achievable using standard methods of crystallization is around 5 µm.5 A useful quality criterion for enzymes is the enzymatic activity, which quantifies the ability of the enzyme to catalyze a specific reaction. As the catalytic properties are the main reason for manufacturing enzymes, any process must have a minimal detrimental effect on the activity. Protein yield can be quantified in terms of the total activity, and purity can be measured in terms of the specific activity, which is the total activity normalized to the total protein content. The specific activity thus accounts for both impurity proteins and deactivated enzyme.25,26
2. Experimental Procedures 2.1. Materials. All chemicals used for the sample assay and buffer preparation were of reagent grade or higher. JBM was purchased from A. L. Jowitt, 612 Runaway Bay Drive, Bridgeport Texas 76426 USA (screen #40) and was used “as is”, without further preparation. Phosphate buffers were prepared by mixing appropriately concentrated aqueous potassium dihydrogen phosphate and dipotassium hydrogen phosphate solutions to reach the required pH. For the purification experiments food-quality NaCl (kindly donated by ESCO Bernburg, screen 3.2–1.5 mm), reagent grade Li2SO4 · H2O (Sigma-Aldrich, product number 398152) and technical poly(ethylene glycol)s with average molar masses of 4000 and 6000 g/mol (kindly donated by Sasol Germany GmbH, product names “Lipoxol 4000 and Lipoxol 6000”, lot numbers 06/1 and 05/20, respectively) were used. High activity urease was purchased from Sigma-Aldrich (g600 U/mg, product number U0251). 2.2. Assays. An optical microscope (Olympus, model BH2-UMA coupled with a Sony CCD-camera) was used to characterize the precipitates from the protein extracts at magnifications of ×134, ×335, and ×670. Images were recorded on a PC using the software DTAAcquire (Data Translation, Inc.). Protein concentration was measured photometrically at λ ) 595 nm using the Bradford method with bovine serum albumin as reference. Using this method only dissolVed protein is measured. The enzymatic activity of urease solutions was determined by measuring the amount of ammonia produced by the decomposition of urea using the phenol-hypochlorite reaction (Berthelot color reaction), which is described, among others, by Weatherburn.27 Sediments and precipitates obtained from 10 g of JBM were dissolved in deionized water at room temperature and then diluted if required. 200 µL of 125 mM urea solution prepared with phosphate buffer at pH 6.5 were added to 50 µL of the urease solution, and the reaction was terminated by adding 1 mL of a phenol solution after 3, 9, and 15 min, respectively. Thereafter, 750 µL of a 0.9% v/v hypochlorite solution was added. After 20 min the extinction of the samples was measured photometrically at λ ) 630 nm. As the urease activity depends on pH and temperature, it is important to keep these parameters constant.28 Here, the test is carried out at 37 °C and pH ) 6.5. The activity of urease is determined from the slope of a plot of the extinction against time. One unit (U) corresponds to the cleavage of 1 µmol of urea per minute and is approximately equivalent to 0.33 Sumner units.29 It is known that the reaction product (ammonia ions) as well as metal ions affect the urease activity.5 Since all measurements were carried out under identical conditions, any effect due to contaminants or reactant concentration can be neglected in the comparison of the data. The dependence of the absorbance on the decomposition time was found to be linear, and no loss of urease activity was detected in the time span of the experiments. Sodium dodecyl sulfate-poly(acrylamide) gel electrophoresis (SDS-PAGE) was used to identify individual proteins present at various stages of the process. A 12.5% T poly(acrylamide) gel was used, and the proteins were rendered visible with a Coomassie blue stain. The PageRuler molecular mass marker (Fermentas, PageRuler Protein Ladder #SM0661) was employed for molecular mass determination (Figure 1, lane 1). 2.3. Procedure. The purification process consists of two main steps: extraction and cooling crystallization. Ideally, the extraction step should selectively extract the target protein only, which is then precipitated in
Weber et al.
Figure 1. SDS-PAGE. The bands (a) and (b) (right) are due to urease and precanavalin, respectively, the molecular mass of the marker proteins (lane 1) is indicated to the left. Additional lanes: 2: JBM proteins extracted with 50 mM phosphate buffer at pH 7.0, 3: commercial urease, 4: urease crystals obtained using acetone and 2-mercaptoethanol (see ref19), 5: precipitate obtained with 7.5% PEG 4000 at pH 6.0 without 2-mercaptoethnol, 6–8: precipitate obtained with 7.5% PEG 4000 with 50 mM 2-mercaptoethanol at pH 5.5, 6.0 and 6.5, respectively, 9–11: precipitate obtained with 7.5% PEG 6000 without 2-mercaptoethanol at pH 5.5, 6.0 and 6.5, respectively. the second process step. In reality one can merely expect to achieve a relative increase of the target protein concentration with respect to the total protein content of the raw material in the extraction step. In this case the precipitation step must lead to a further purification by selectively precipitating the target protein. Here, the desired protein is urease. In addition to the target protein, jack beans contain a significant amount of other proteins, including canavalin (Smith et al.30), concanavalin A (Olson and Liener31), concanavalin B (Hennig et al.32) among others, which have to be separated either in the initial extraction step or in the subsequent crystallization step, where urease crystallizes and other proteins remain in the extract. After each step, any solid and liquid phases are separated by centrifugation at 8600 g for 20 min and at a constant temperature. Extraction was carried out in a temperature controlled 100 mL beaker. Ten grams of JBM were suspended in 50 mL of solvent at 28 °C. The solvents employed were either phosphate buffer solutions with different concentrations of poly(ethylene glycol) with a molar mass of either 4000 or 6000 g/mol and different pH values (for PEG 4000 the influence of the presence of 2-mercaptoethanol was also investigated) or aqueous solutions of Li2SO4 and NaCl. The protein solution was stirred for up to 7 min using a motorized overhead stirrer operating at 800 rpm equipped with a paddle impeller. Cooling crystallization was carried out in a refrigerator at 4 °C without agitation and using a natural cooling profile. Seeding was not employed. After two days any precipitate formed was separated from the solution, and both liquid and solid phases were characterized.
3. Results and Discussion Previous work19 confirmed that crystallization based upon the method of Sumner is indeed a very effective separation technique. However, due to the limitations resulting from both the complex composition of the raw material and the chemical properties of acetone this procedure can only be improved marginally by further purification steps. The main limitation of this method is low extraction effectiveness. Our work showed that a maximum of 40% of the urease present is extracted in the presence of acetone. On the other hand, the use of acetone in the extraction solvent provides for good selectivity due to effective separation of insoluble precanavalin. The investigations described in the following aim at establishing whether alternative extraction solvents and precipitants can improve the extraction yield of urease while maintaining the selectivity of the established method. For the purpose of comparison the purest commercially available urease, i.e. that with the highest specific activity (Sigma-Aldrich, purchase number U0251) was also investigated.
Crystallization as Purification for Jack Bean Urease
Crystal Growth & Design, Vol. 8, No. 2, 2008 713
Table 1. Experimental Parameters of the Experiments Reported Here and Crystallization Results extraction solution
pH
crystallization temperature/°C
PEG 6000, 6–8 0–12.5 w/v %
4–22
PEG 4000, 0–20 w/v %
5–7
4
Li2SO4, 0–30 w/v % in water NaCl, 0–35 w/v % in water
not adjusted
4
not adjusted
4
results well-shaped particles
