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ORIGINAL RESEARCH PAPER

Advanced protein crystallization using water-soluble ionic liquids as crystallization additives

Dariusch Hekmat Æ Dirk Hebel Æ Sebastian Joswig Æ Michael Schmidt Æ Dirk Weuster-Botz

Received: 1 May 2007 / Accepted: 16 June 2007 / Published online: 1 August 2007

� Springer Science+Business Media B.V. 2007

Abstract The application of five water-soluble,

halogen-free, alkylammonium-based ionic liquids

(ILs) as additives for advanced crystallization of

lysozyme was investigated. Their biocompatibility

was determined by long-term measurement of the

overall mean relative enzyme activities. These were

maximally reduced by about 10–15% when up to

200 g IL l�1 was added. Sitting-drop vapor diffusion

crystallization experiments revealed that the addition

of some of the ILs led to less crystal polymorphism

and precipitation was avoided reliably even at larger

NaCl concentrations. The addition of ILs tended to

result in larger crystals. The kinetics of lysozyme

crystallization were significantly enhanced using ILs

as crystallization additives, e.g. by a factor of 5.5

when 100 g ethanolammonium formate l�1 was

added. ILs with ‘‘soft’’ anions, such as formate or

glycolate, were superior to ILs with ‘‘hard’’ anions,

like nitrate.

Keywords Advanced crystallization � Crystallization additives � Crystallization kinetics � Water-soluble Ionic liquids � Lysozyme

Introduction

The application of crystallization as a purification

step of proteins is economically attractive because the

number of required cost-intensive chromatography

steps can be reduced. Furthermore, crystallized

proteins may offer superior properties compared to

liquid formulations or amorphous lyophilisates due to

better handling, longer shelf-life, and higher purity

(Schmidt et al. 2004). Furthermore, crystalline phar-

maceutical proteins may provide the advantageous

possibility of controlled slow release of activity (Basu

et al. 2004). The empirical crystallization methods for

structural analysis of proteins have been applied for

decades and, consequently, optimized techniques

have emerged (Bergfors 1999). However, due to the

complexity of the involved systems and the lack of

transferability of experimental findings, the crystal-

lization step still represents a bottleneck in many

cases. In fact, many proteins are difficult to crystal-

lize or do not crystallize at all. Here, very little

systematic knowledge exists. As a consequence,

existing crystallization processes are typically slow

and the yields as well as the reproducibilities are

often low. Therefore, a new strategy was sought in

order to advance the crystallization process. Here, the

application of biocompatible, water-soluble ionic

liquids (ILs) as crystallization additives may be

promising. However, such a strategy has not been

investigated much in the past. ILs are organic salts

which are liquid at room temperature and virtually

D. Hekmat (&) � D. Hebel � S. Joswig � M. Schmidt � D. Weuster-Botz Institute of Biochemical Engineering, Munich University

of Technology, Boltzmannstr. 15, 85748 Garching,

Germany

e-mail: [email protected]

123

Biotechnol Lett (2007) 29:1703–1711

DOI 10.1007/s10529-007-9456-9

have zero vapor pressure (Seddon 1997). ILs already

have proven their advantageous properties as novel

solvents in a number of applications (Wasserscheid

and Welton 2002; Song 2004; Yang and Pan 2005).

The toxicity of ILs has been investigated using

different methods (Ranke et al. 2004; Swatloski et al.

2004) and some biocompatible ILs have been iden-

tified. The first work on protein crystallization using

ILs as additives was reported by Garlitz et al. (1999).

Lysozyme crystallized easier using about 40–50 g

ethylammonium nitrate l�1 compared to pure aqueous

solutions. Furthermore, enzymes retained their activ-

ity and had a higher stability in aqueous solutions with

addition of ILs. Additionally, ILs were a useful

additive for improving the monodispersity of those

types of proteins which exhibit multiple aggregation

states. According to Seddon (1997), ILs are able to

suppress conventional solvation and solvolysis phe-

nomena. However, the nature of solvation in ILs is

complex. Although the process of hydrogen-bonding

in molecular and ionic solvents is mechanically

similar, the presence of proximal counter ions com-

plicates the analysis of specific interactions (Znamen-

skiy and Kobrak 2004). Therefore, more detailed

studies are necessary to be able to reflect the possible

enzyme-solvent interactions and to provide structural

information about how the ILs interact with the

protein and replace the interactions of water (Song

2004; Yang and Pan 2005). Obviously, there is a large

demand for basic research on a molecular level in this

field which is actually under way worldwide. On the

other hand, enough experience already exists in order

to simultaneously perform applied research studying

the crystallization process with ILs on a macroscopic

basis. This is the aim of the present work, to

investigate the impact of biocompatible, water-solu-

ble ILs as crystallization additives in order to advance

the crystallization process. Lysozyme was chosen as

the exemplary protein.

Materials and methods

Ionic liquids (ILs), protein, microorganism, and

chemicals

Five different ILs were used in the present study

which were all easily water-soluble. Some properties

and the chemical structures of the ILs are given in

Table 1. IL1.1, IL1.2, and IL4 were supplied by

IoLiTec GmbH & Co. KG, Denzlingen, Germany.

IL2 and IL3 were supplied by Bioniqs Ltd., York,

Great Britain. IL1.1 and IL1.2 have a primary

alkylammonium cation, IL2 has a secondary alky-

lammonium cation, IL3 has a tertiary alkylammonium

cation, and IL4 has a quaternary alkylammonium

cation. Lysozyme from chicken egg white was

purchased from Sigma-Aldrich, Taufkirchen, Germany

(No. 62971). Micrococcus lysodeicticus, used for the

lysozyme activity test, was from Sigma-Aldrich. All

other chemicals were analytical grade purchased from

Merck, Darmstadt, Germany.

Determination of the maximum lysozyme

solubility in water and in buffered

IL-water solutions

Equilibrated saturated solutions of lysozyme in

demineralized water with and without acetate buffer

and in buffered aqueous solutions with 100 g ILs l�1

were made using a laboratory rotator (neoLab GmbH,

Heidelberg, Germany) at 5 rev min�1 for 1 h. These

solutions were centrifuged at 13,000 g for 3 min. The

UV absorbance of the supernatant was measured at

280 nm and compared to a calibration standard.

Determination of the activity of lysozyme in

aqueous solutions with and without ILs

The lysozyme activity test is based on the ability of

the enzyme to break down the cell wall of Micro-

coccus lysodeicticus via hydrolyzation of the b-1,4 linkages between N-acetylmuramic acid and N-ace-

tyl-D-glucosamine residues in peptidoglycan and

between N-acetyl-D-glucosamine residues in chitod-

extrin. Gram-positive cells like Micrococcus are quite

susceptible to this hydrolysis since their cell walls

have a high proportion of peptidoglycan. Suspensions

of 2.8 mg Micrococcus lysodeicticus l�1 in 1 M

phosphate buffer, a pH 5.5, were prepared. Sample

solutions with 100 mM acetate buffer, pH = 5.5,

were made with a lysozyme at 1 g l�1 using a

laboratory rotator at 20 rev min�1 for 2 h. The

activity measurements were performed in a 96-well

optical bottom, microtiter plate (Nunc GmbH & Co.

KG, Wiesbaden, Germany) using a thermostatable

ultra-microplate reader. The operating temperature

was set to 33 ± 0.1�C. Relative activities of lysozyme

1704 Biotechnol Lett (2007) 29:1703–1711

123

solutions were calculated from the quotients of the

measured absolute protein activities of the IL-water

solution and the IL-free aqueous solution at a given

time. The concentrations of the ILs were varied

between 50 and 200 g l�1.

Sitting-drop vapor-diffusion crystallization

experiments

The sitting-drop crystallization experiments were

performed at room temperature in 24-well Chry-

schem plates type HR3-158 (Hampton Research

Corp., Aliso Viejo, CA, USA) sealed with Crystal

Clear sealing tape (Manco Inc., Avon, OH, USA).

The sitting-drop tray had a diameter of about 6 mm

and a maximum filling volume of about 40 ll. For each tray, 10 ll lysozyme solution was mixed with 10 ll respective reservoir solution containing buffer and crystallization additives. The lysozyme concen-

tration was altered between 10 and 50 g l�1. The

concentration of the conventional antisolvent NaCl

was between 20 and 50 g l�1 and the concentration

of the ILs was from 25 to 100 g l�1. All concentra-

tion data refer to the start concentrations in the trays.

An acetate buffer with a pH of 4 was used. Due to the

different basicity of IL1.1, IL2, and IL3, the pH in the

Table 1 List of the used alkylammonium-based halogen-free water-soluble ionic liquids (ILs)

Type of IL, properties of pure IL Chemical structure

Ethanolammonium formate (IL1.1), MW = 107 g mol�1, pH = 8.5 MP = �82�C

Ethylammonium nitrate (IL1.2), MW = 108 g mol�1, pH = 4.3 MP = 13�C

Bis(2-methoxyethyl)ammonium acetate (IL2), MW = 193 g mol�1, pH = 8.2 MP < �20�C

N,N-dimethylethanolammonium glycolate (IL3), MW = 165 g mol�1, pH = 8.4, MP < �20�C

Choline dihydrogenphosphatea (IL4), MW = 201 g mol�1, pH = 6.6 (at cIL4 = 80%), MP = 119�C

a Choline dihydrogenphosphate (IL4) is a so-called near-IL; liquid at room temperature when diluted with 20% (v/v) H2O

Biotechnol Lett (2007) 29:1703–1711 1705

123

respective sitting-drops varied from 6.2 to 7.0.

Microphotographs of the sitting-drops were made

using a microscope type Zeiss Axioplan (Carl Zeiss

MicroImaging GmbH, Göttingen, Germany) and a

digital camera type DSC-S75 (Sony Deutschland

GmbH, Köln). The experimental set-up was designed

in order to prevent shocks and/or vibrations acting

upon the crystallization plates.

Determination of crystallization kinetics

The crystallization kinetics were obtained via the

determination of the total cross-sectional crystal

surface of the sitting-drop microphotographs as a

function of time. For this purpose, a public domain

image processing software was used (ImageJ, version

1.37v, http://rsb.info.nih.gov/ij). Crystallization equi-

librium was reached when the measured cross-

sectional crystal surface was at maximum and

constant. The normalized total cross-sectional crystal

surface was calculated from the quotient of the actual

value divided by the maximum total cross-sectional

crystal surface at equilibrium. For comparison of the

crystallization kinetics, a parameter t90 was intro-

duced which was defined as the time to reach 90 % of

the measured maximum cross-sectional crystal sur-

face at equilibrium.

Results and discussion

Determination of the maximum lysozyme

solubility in water and in buffered IL-water

solutions

The results of the lysozyme solubility measurements

are presented in Table 2. As expected, solubility was

highest in pure water. The addition of acetate buffer

reduced the solubility markedly. The solubility of

lysozyme dropped significantly when 100 g l�1 ILs

were added to the aqueous solutions. The reduction of

solubility was least with IL4. Interestingly, for IL1.1

and IL3, the decrease to 28–30 g l�1 was in the same

order of magnitude of an aqueous lysozyme solution

with about 20–25 g NaCl l�1. With 100 g IL1.2 l�1,

the solubility of lysozyme was very low. The

solubility measurements without ILs were well in

accordance with data of Howard et al. (1988).

Determination of the activity of lysozyme in

aqueous solutions with and without ILs

The time-courses of the activity of lysozyme dis-

solved in water without ILs was monitored during

time periods of 56–70 days. As can be seen from

Fig. 1, the activity data scattered noticeable. The

solid lines represent linear fits to the experimental

data. The activity of the dissolved lysozyme stored at

4�C dropped rather fast initially. Then, the negative slope was relatively small and about 70% of the

initial activity was reached after 70 days. In contrast

Table 2 Determination of the maximum lysozyme solubility in water and in buffered IL-water solutions. The mean mea-

surement error was 5%

Composition of solution pH Solubility

[g l�1]

H2O demin. 3.4 415

100 mM acetate buffer 4.0 382

100 mM acetate buffer 5.5 377

50 mM acetate buffer 5.5 270

100 g l�1 ethanolammonium formate (IL1.1)a

7.0 30

100 g l�1 ethylammonium nitrate (IL1.2)a 5.4 4

100 g l�1 bis(2-methoxyethyl)ammonium acetate (IL2)a

6.2 74

100 g l�1 N,N-dimethylethanolammonium glycolate (IL3)a

6.2 28

100 g l�1 choline dihydrogenphosphate (IL4)a

5.2 99

a 50 mM acetate buffer

0

20

40

60

80

100

0 10 20 30 40 50 60 70

Time [d]

P ro

te in

a ct

iv ity

[% ]

Fig. 1 Time-courses of the activity of lysozyme dissolved in water without IL. cLysozyme = 1 g l

�1, 100 mM acetate buffer, pH = 5.5. Storage of solutions at 4�C (h) and 21�C (d). The solidlines represent linear fits to the experimental data

1706 Biotechnol Lett (2007) 29:1703–1711

123

to this, the time course of the activity of the dissolved

lysozyme stored at 21�C was approximately linear from the beginning on, however, the negative slope

was comparatively larger. Hence, about 70% of the

initial activity was reached after 56 days. This

temperature dependent behavior was expected since

the lower storage temperature preserved the enzyme

activity. The relative lysozyme activities of the

solutions containing ILs are given in Table 3. As

can be seen, the overall mean relative enzyme

activities over time were reduced by about 10–15%

for IL1.1, IL1.2, IL2, and IL3. On the other hand, the

addition of 125–200 g l�1 of IL4 apparently led to a

slight increase of enzyme activity due to possible

renaturating effects. Such behavior has been

described in the literature (Summers and Flowers

2000; Lange et al. 2005). The results indicate that

the chosen alkylammonium-based water-soluble

ILs were well suitable for the present application

involving biologically active proteins.

Sitting-drop vapor-diffusion crystallization

experiments

At first, sitting-drop crystallization experiments of

lysozyme without the addition of ILs were performed

at variable protein and NaCl concentrations. At

medium concentrations of about 20–40 g l�1 for

both protein and NaCl, tetragonal crystals were

obtained in most cases. However, at NaCl concen-

trations above 40 g l�1, in addition to the tetragonal

lysozyme crystals, precipitation occurred frequently

and sea urchin-like formations consisting of mono-

clinic crystal needles structured around amorphous

aggregates appeared sometimes (see Fig.2A). This

polymorphism was described earlier by Muschol and

Rosenberger (1997). Next, sitting-drop crystallization

experiments of lysozyme with the addition of IL3

were performed. The IL3 concentration was varied

between 25 and 100 g l�1 and the NaCl concentration

was in the range of 30–50 g l�1. Independently of the

varying concentrations of IL3 and NaCl, tetragonal-

like shaped crystals were obtained. Interestingly, no

precipitation and no polymorphism was observed

anymore even at higher NaCl concentrations. Similar

results were obtained with IL1.1 (see Fig. 2B), IL2,

and IL4. In contrast to this, the addition of IL1.2 led

to spontaneous precipitation followed by slow partial

or total transformation of precipitate into fascicular

structures of monoclinic crystals (see Fig. 2C).

Furthermore, the experiments showed that the addi-

tion of ILs tended to result in larger crystals. This is

demonstrated in Fig. 2D using IL4 as crystallization

additive. Here, the largest crystal length was 1.6 mm.

The overall largest crystal length of 1.7 mm was

achieved with 25 g IL1.1 l�1 after 50 h while the

largest crystal length without addition of ILs of

1.1 mm was achieved after 48 h.

Determination of crystallization kinetics

During the experiments for the determination of the

crystallization kinetics, the protein and NaCl concen-

trations were kept constant at 50 g l�1 and 25 g l�1,

respectively. Fig. 3 shows typical microphotographs of

tetragonal-like shaped lysozyme crystals during tran-

sient growth in sitting-drops using IL3 as crystallization

Table 3 Mean relative activities over time of lysozyme in IL- water solutions. The overall mean measurement error was 12%

Type of IL,

concentration

of IL [g l�1]

Mean relative

activity [%]

Overall mean relative

activity [%]

Ethanolammonium formatea(IL1.1)

50 94 91

125 94

200 85

Ethylammonium nitrateb (IL1.2)

50 94 91

125 91

200 88

Bis(2-methoxyethyl)ammonium acetatea (IL2)

50 89 85

125 85

200 81

N,N-dimethylethanolammonium glycolatea (IL3)

50 92 90

125 94

200 84

Choline dihydrogenphosphateb (IL4)

50 92 102

125 103

200 109

a Storage of solutions at 4�C, monitored during a time period of 70 days b Storage of solutions at 21�C, monitored during a time period of 56 days

Biotechnol Lett (2007) 29:1703–1711 1707

123

additive. As can be seen, the growth of distinct

lysozyme crystals could be followed easily and the

cross sectional crystal surface as a function of time

could be obtained via image processing. Exemplary

results for two independent experiments using IL1.1 are

presented in Fig. 4. Due to the basic nature of IL1.1, the

Fig. 2 Microphotographs of lysozyme crystals with different morphologies at equilibrium in sitting-drops with 50 mM

acetate buffer. (A) No IL, cLysozyme = 10 g l �1, cNaCl =

45 g l�1, pH = 4.0, tetragonal crystals and sea urchin-like formations consisting of monoclinic crystal needles structured

around amorphous aggregates. (B) 62.5 g ethanolammonium

formate (IL1.1) g l�1, cLysozyme = 50 g l �1, cNaCl = 25 g l

�1, pH = 6.7, tetragonal crystals. (C) 100 g ethylammonium nitrate l�1 (IL1.2), cLysozyme = 50 g l

�1, cNaCl = 25 g l �1,

pH = 5.4, fascicular structure of monoclinic crystals. (D) 25 g l�1 choline dihydrogenphosphate (IL4), cLysozyme = 50 g l�1, cNaCl = 25 g l

�1, pH = 5.2, tetragonal crystals

Fig. 3 Exemplary microphotographs of

tetragonal-like shaped

lysozyme crystals during

transient growth in sitting-

drops used for the

determination of the

crystallization kinetics.

100 g N,N- dimethylethanolammonium

glycolate (IL3) l�1, cLysozyme = 50 g l

�1, cNaCl = 25 g l

�1, 50 mM acetate buffer, pH = 6.2.

(A) 1 h after start of experiment. (B) after 2 h (C) after 3 h (D) after 4 h

1708 Biotechnol Lett (2007) 29:1703–1711

123

pH in the sitting-drops varied from 6.3 to 7.0. The

crystallization kinetics were significantly increased by

10–18-fold with rising concentrations of the IL from 25–

100 g l�1. Crystallization equilibrium using 100 g l�1

of IL1.1 was reached as early as 5–8 h after the start.

Clearly, reproducibility was poor. However, vapor-

diffusion experiments, in general, do not yield good

reproducibilities.

Figure 5 gives a comparison of the time-courses of

the normalized total cross-sectional crystal surface of

sitting-drops with and without the addition of ILs as

crystallization additives. The fastest crystallization

kinetics without the addition of ILs was measured

using 30 g lysozyme l�1, 40 g NaCl l�1, and 50 mM

acetate buffer at pH 4.0. The pH in the sitting-drop

experiments with ILs using 50 mM acetate buffer

C)B)

lanoitce s-ssor c lato t dez ila m ro

N ecafrus latsyrc

0 2 4 6 8 10 Time [h]

0 2 4 6 8 10 Time [h]

0.0

0.2

0.4

0.6

0.8

1.0

0 2 4 6 8 10 Time [h]

A)

Fig. 4 Exemplary time-courses of the normalized total cross- sectional crystal surface of sitting-drops using ethanolammo-

nium formate (IL1.1). cLysozyme = 50 g l �1, cNaCl = 25 g l

�1, 50 mM acetate buffer, pH = 6.3–7.0. Two independent exper-

iments for each concentration level of IL. The required time to

reach crystallization equilibrium is given in brackets. (A) 25 g l�1 h (50 h); d (146 h). (B) 62.5 g l�1 h (26 h); d (50 h). (C) 100 g l�1 h (5 h); d (8 h). The mean measurement error was 10%

lanoitc es-ssorc latot dezil a mr o

N ecafrus latsyrc

B)

lanoitces-ssorc latot dezila mro

N ecafrus latsyrc

0.0

0.2

0.4

0.6

0.8

1.0 A)

0.0

0.2

0.4

0.6

0.8

1.0

0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35

C) D)

Fig. 5 Comparison of the time-courses of the

normalized total cross-

sectional crystal surface of

sitting-drops with 50 mM

acetate buffer with ILs (h;

cLysozyme = 50 g l �1,

cNaCl = 25 g l �1) and

without ILs (d;

cLysozyme = 30 g l �1,

cNaCl = 40 g l �1, pH = 4.0).

The required time to reach

crystallization equilibrium

is given in brackets. (A) 100 g l�1 IL1.1, pH = 7.0 (5 h). (B) 100 g l�1 IL2, pH = 6.2 (50 h). (C) 100 g l�1 IL3, pH = 6.2 (124 h). (D) 100 g l�1 IL4, pH = 5.2 (66 h). The mean

measurement error was

10%

Biotechnol Lett (2007) 29:1703–1711 1709

123

varied from 5.2 to 7 due to the different basicity of

the ILs. The fastest crystallization kinetics were

observed with IL1.1 followed by IL3 and IL2, each

with concentrations of 100 g l�1. The crystallization

kinetics with IL4 were the slowest. For quantification,

the parameter t90 was calculated from the experi-

mental data. The results are presented in Table 4. The

t90-reference value without addition of ILs was 22 h.

The strongest advancement of the crystallization

kinetics using ILs was achieved with 100 g IL1.1 l�1

where a 5.5-fold increase was observed. The t90-value

for IL1.2 was not available because image processing

of the fascicular structures was not possible. The

addition of 100 g IL2 l�1 resulted in a 29% increase

in crystal growth kinetics. The second strongest

increase of the crystallization kinetics by 4.4-fold was

achieved with 100 g IL3 l�1. Finally, a 31% decel-

eration of the crystallization kinetics was measured

using 100 g IL4 l�1. This deceleration, however,

yielded comparatively larger crystals.

The results indicate that the kinetics of lysozyme

crystallization were significantly enhanced by addi-

tion of water-soluble ionic liquids. ILs with ‘‘soft’’

anions like formate or glycolate were superior to ILs

with ‘‘hard’’ anions like nitrate. Precipitation could be

avoided reliably even at larger salt concentrations and

crystal polymorphism was reduced compared to

experiments without ionic liquids. Furthermore, the

addition of ILs tended to result in larger crystals.

Future investigations have to be performed in order to

evaluate the removal of ionic liquids from the protein

crystals. Here, according to Cvetkovic et al. (2005),

the diffusive removal by simple washing of the

crystals seems to be a possible promising way.

Acknowledgements The authors thank Lorenz Chatwell and Arne Skerra for valuable advices in the initial phase of the

work. The authors also acknowledge the experimental

contribution of Hannes Schmid.

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Table 4 Summary of the kinetics of lysozyme crystallization with and without ILs. t90 was defined as the time to reach 90% of the measured maximum cross-sectional crystal surface at equilibrium

Type of IL, experimental conditions t90 [h] Degree of enhancement [%]

No IL, cLysozyme = 30 g l �1, cNaCl = 40 g l

�1, 50 mM acetate buffer, pH = 4.0 22 100

100 g l�1 ethanolammonium formate (IL1.1), pH = 7.0a 4 550

100 g l�1 ethylammonium nitrate (IL1.2), pH = 5.4a n.a.b –

100 g l�1 bis(2-methoxyethyl)ammonium acetate (IL2), pH = 6.2a 17 129

100 g l�1 N,N-dimethylethanolammonium glycolate (IL3), pH = 6.2a 5 440

100 g l�1 choline dihydrogenphosphate (IL4), pH = 5.2a 32 69

a cLysozyme = 50 g l �1, cNaCl = 25 g l

�1, 50 mM acetate buffer b t90-value not available. Spontaneous precipitation was observed followed by slow partial or total transformation of precipitate into

fascicular structures of monoclinic crystals

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