preparation, optimization, and structures of cross-linked enzyme aggregates (cleas)

Preparation, Optimization, and Structures ofCross-Linked Enzyme Aggregates (CLEAs)

R. Schoevaart,1,3 M.W. Wolbers,2 M. Golubovic,2 M. Ottens,2

A.P.G. Kieboom,3 F. van Rantwijk,1 L.A.M. van der Wielen,2 R.A. Sheldon1

1Biocatalysis and Organic Chemistry, Department of Biotechnology, DelftUniversity of Technology, Julianalaan 136, 2628 BL Delft, The Netherlands;telephone: + 31 15 2782683; fax: + 31 15 2781415;e-mail: [email protected] Technology, Department of Biotechnology, Delft University ofTechnology, Julianalaan 67, 2628 BC Delft, The Netherlands3Industrial Fermentative Chemistry, Leiden University, P.O. Box 9502,2300 RA Leiden, The Netherlands

Received 17 December 2003; accepted 11 May 2004

Published online 18 August 2004 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bit.20184

Abstract: The broad applicability of the cross-linking ofenzyme aggregates to the effective immobilisation of en-zymes is demonstrated and the influence of many parame-ters on the properties of the resulting CLEAs is determined.The relative simplicity of the operation ideally lends itself tohigh-throughput methodologies. The aggregation methodwas improved up to 100% activity yield for any enzyme.For the first time, the physical structures of CLEAs areelucidated. B 2004 Wiley Periodicals, Inc.

Keywords: immobilization; CLEA; CLEC; protein

INTRODUCTION

A substantial effort has been devoted (Cao et al., 2003;

Kragl, 1996; Messing, 1975) to developing effective immo-

bilization techniques to increase the operational stability of

enzymes and to facilitate their recovery and recycling. Im-

mobilization methods basically can be divided into two

types: binding to or inclusion in a support (Boller et al.,

2002) or cross-linking of pure protein (Cao et al., 2000). A

distinct disadvantage of carrier-bound enzymes is the dilu-

tion of catalytic activity resulting from the introduction of a

large proportion of noncatalytic mass, generally ranging

from 90–> 99% of the total mass. This inevitably leads to

lower volumetric and space-time yields and lower catalyst

productivities. Immobilization via cross-linking of enzyme

molecules with bifunctional cross-linking agents, in con-

trast, does not suffer from this latter disadvantage. Since the

molecular weight of the cross-linking agent is negligible

compared with that of the enzyme, the resulting biocatalyst

essentially comprises 100 wt% protein.

The technique of protein cross-linking via the reaction of

glutaraldehyde with reactive amine residues on the protein

surface was initially developed in the 1960s (Doscher and

Richards, 1963). However, this method of producing cross-

linked enzymes (CLEs) had several drawbacks, such as low

activity retention, poor reproducibility, low mechanical

stability, and difficulties in handling the gelatinous CLEs.

The cross-linking of a crystalline enzyme was first (Quiocho

and Richards, 1964) applied to stabilize enzyme crystals for

X-ray diffraction studies. Subsequently, these cross-linked

enzyme crystals (CLECR) were successfully commercial-

ized into industrial biocatalysts (Haring and Schreier, 1999;

Lalonde, 1997; Margolin, 1996; Margolin and Navia, 2001;

St. Clair and Navia, 1992). An inherent disadvantage of

CLECs is the need to crystallize the enzyme, which is often

a laborious procedure requiring enzyme of high purity. It

was shown (Cao et al., 2001) that comparable results could

be achieved by precipitating the enzyme and cross-linking

the resulting physical aggregates. This led to the develop-

ment of a new form of immobilized enzymes: cross-linked

enzyme aggregates (CLEAR).

Aggregation and precipitation induced by the addition

of salts, organic solvents, non-ionic polymers or acids

(Hofland et al., 2000) to aqueous solutions of proteins is a

commonly used method of protein purification. These

physical aggregates are supramolecular structures held

together by noncovalent bonding and redissolve when

dispersed in water. Cross-linking produces CLEAs, in which

the structure of the aggregates and, hence, the catalytic

activity of the individual proteins is maintained. Interest-

ingly, activity yields exceeding that of the native enzyme

were observed with lipases, and subsequently were also

observed with other enzymes. This hyperactivation is

thought to find its origin in conformational changes of the

protein induced by the aggregated state (Lopez-Serrano

et al., 2002). Cross-linked enzyme aggregates made from

lipases and penicillin acylase (Van Langen et al., 2002)

illustrated the effect of various parameters, such as the

precipitant and additives like surfactants and crown ethers,

on the activities.

B 2004 Wiley Periodicals, Inc.

Correspondence to: R.A. Sheldon

In this study a high throughput method is presented for

the general preparation and optimization of CLEAs of a

wide variety of enzymes. The method consists of a precip-

itant selection and concentration optimization followed by

optimization of the cross-linker and protein concentration to

open the way to fast and efficient CLEA preparation.

MATERIALS AND METHODS

General

The lipases and trypsin were obtained from Novozymes

(Bagsværd, Denmark); galactose oxidase from Hercules

(Barneveld, The Netherlands); h-galactosidase and phytase

from DSM (Delft, The Netherlands); laccase, alcohol dehy-

drogenase ,and formate dehydrogenase from Julich Fine

Chemicals (Julich, Germany). All other enzymes and chem-

icals were purchased from Sigma. Scanning Electron Mi-

croscope used was a Philips XL 20, samples of CLEAs were

freeze-dried prior to analysis. UV/Vis spectroscopy was

performed on a Varian Cary 3 Bio equipped with a Cary

temperature controller or a Greiner microplate U-FORM

96K microtiter plate reader at room temperature. NMR

experiments were performed with DPX-300 spectrometers

(Bruker, Karlsruhe, Germany) at 75.48 MHz for 13C with

methanol as internal standard (49 ppm) and 5% D2O.

For the assays, an enzyme activity is necessary that in-

duces a change in absorption (�A) of 0.1 to 0.5/min.

Dilutions of the enzymes were made so that the �A per

minute never exceeded 0.5. All activities were correlated

to the native enzyme, taken as 100% (no absolute activities

are given).

Aggregate Activity

To a volume of 10 AL enzyme solution (with a constant

amount of enzyme) 90 AL of precipitant with proper dilution

was added and after 15 minutes 900 AL buffer (100 mM

potassium phosphate, pH 7.3). An aliquot was subsequently

transferred into cuvettes (100 AL/mL) or onto microtiter

plates (20 AL /200 AL). The pH used was the optimal pH

according to the specific enzyme activity test.

CLEA Activity

For the cross-link optimization 90 AL precipitant containing

glutaraldehyde in varying concentrations was added to

10 AL enzyme solution. To the glutaraldehyde stock 1 vol%

phosphoric acid was added and the pH was adjusted to 7.3

with diluted sodium hydroxide prior to mixing with the

precipitants. The samples were incubated at room temper-

ature without shaking. For the volumetric activity optimi-

zation 90 AL precipitant containing glutaraldehyde was

added to 10 AL solution with a varying protein content and

after the specific cross-linking time this mixture was

quenched with 900 AL buffer.

General CLEA Scale-Up

For the preparative production of the different CLEAs a

general procedure is followed. In a 50-mL centrifuge tube

with a magnetic strirrer bar 1 mL enzyme (25 mg) solution

in 100 mM buffer (mentioned in the activity assay) was

added to 9 mL precipitant at room temperature unless stated

otherwise. Then, glutaraldehyde (25%) was added to the

desired end concentration and the suspension was stirred for

2.5 h. The suspension was diluted with 10 mL buffer and

centrifugated. The pellet was resuspended in buffer and

centrifuged again. This procedure was performed two times

in total. Subsequently, the washed CLEA was resuspended

in 1 mL buffer and stored at 4jC.

Lipase

The standard activity test (Lopez-Serrano et al., 2002) was

performed with p-nitrophenyl propionate as substrate

(7.8 mg in 1 mL ethanol; 10 AL per mL assay buffer).

Assay buffer (100 mM potassium phosphate, pH 7.4) con-

tained 0.4 mM p-nitrophenyl propionate at 25jC. The

reaction was monitored at 400 nm. Blank reaction rate: �A

0.00318/min. Additionally, (slow) hydrolysis of triacetine

(2%, V/V) in 50 mM Tris buffer pH 7.4 and 40jC was

performed, monitored by 0.1M NaOH titration. Candida

antarctica lipase A [EC 3.1.1.3] (CaLA) contained 125 units

per mL. The CLEA was prepared in DME with 100 mM

glutaraldehyde and 7 units/mL. Candida antarctica lipase B

[EC 3.1.1.3] (CaLB) contained 308 units and 10 mg protein

per mL. The CLEA was prepared in DME with 150 mM

glutaraldehyde and 15 units/mL. Thermomyces lanuginosus

lipase [EC 3.1.1.3] (TlL) contained 66.7 units/mL. The

CLEA was prepared in tert-butyl alcohol with 100 mM

glutaraldehyde and 5 units/mL. Rhizomucor miehei lipase

[EC 3.1.1.3] (RmL) contained 44.1 units/mL. The CLEA

was prepared in tert-butyl alcohol with 100 mM glutar-

aldehyde and 3 units/mL.

Laccase

Enzymatic oxidation (Bourbonnais and Paice, 1990) of

ABTS (0.2 mg/mL assay) was performed in 0.1M acetate

pH 4.5 and monitored at 420 nm and 25jC. The assay buffer

was saturated with oxygen prior to addition to the cuvet.

Coriolus versicolor laccase [EC 1.10.3.2] (Lacc) contained

0.66 units per mg. The CLEA was prepared in PEG with

100 mM glutaraldehyde and 2 units/mL.

Phytase

Hydrolysis of p-nitrophenyl phosphate (0.4 mM) in 0.1M

acetate pH 4.5 monitored at 400 nm gave the activities of

phytase (Dvorakova et al., 1997). The blank reaction at pH

4.5 was negligible. Aspergillus niger phytase [EC 3.1.3.8]

(Phyt) contained 47 units per mg. The CLEA was pre-

SCHOEVAART ET AL.: PREPARATION, OPTIMIZATION, AND STRUCTURES OF CLEAS 755

pared in ethyllactaat with 100 mM glutaraldehyde and

120 units/mL.

Galactose Oxidase

A solution containing galactose (200 mM, prepared 1 day in

advance to allow for mutarotation), O-toluidine (1.75 mM),

peroxidase (60 U/mL) and 100 mM potassium phosphate at

pH 7.3 was used to detect galactose oxidase activity. The

oxidation of O-toluidine (Avigad et al., 1962) was moni-

tored at 425 nm. Dactylium dendroides galactose oxidase

[EC 1.1.3.9] (GalOx) contained 3000 units per mL. The

CLEA was prepared in tert-butyl alcohol with 100 mM

glutaraldehyde and 225 units/mL.

Trypsin

The activity (Asaad and Engberts, 2003) was monitored

using the same test as for the lipases (hydrolysis of

p-nitrophenyl propionate). Porcine trypsin contained 30%

protein per mg. The CLEA was prepared in saturated am-

monium sulfate with 100 mM glutaraldehyde and 2 mg/mL.

Glucose Oxidase

A solution containing glucose (200 mM, prepared 1 day in

advance to allow for mutarotation), O-toluidine (1.75 mM),

peroxidase (60 U/mL), and 100 mM potassium phosphate at

pH 7.3 was used to detect glucose oxidase activity. The

oxidation of O-toluidine was monitored at 425 nm.

Aspergillus niger glucose oxidase [EC 1.1.3.4] (GlcOx)

contained 50 units and 20% protein per mg. The CLEA was

prepared in saturated ethyllactate with 100 mM glutaralde-

hyde and 125 units/mL.

h-Galactosidase

The assay buffer (potassium phosphate, 100 mM pH 7.3)

contained 0.4 mM p-nitrophenyl-h-D-galactopyranoside

for which hydrolysis (Kim et al., 2003.) is monitored at

25jC and 400 nm. Aspergillus oryzae h-galactosidase [EC

3.2.1.23] (Gal-ase) contained 227 units per mg. The CLEA

was prepared in 2-propanol with 100 mM glutaraldehyde

and 850 units/mL.

ADH

Enzyme activity (Rella et al., 1987) was monitored at 30jC

and 340 nm (Mol ext. = 6.22 � mM�1cm�1) with p-chloro-

acetophenone (1.5 mM) as a substrate in 100 mM potassium

phosphate buffer pH 6.0 and 0.25 mM NADH. Rhodococcus

erythropolis alcohol dehydrogenase [EC 1.1.1.2] (ADH)

contained 77 units and 16.1 mg protein per mL. The CLEA

was prepared in saturated ammonium sulfate with 8 mM

glutaraldehyde at 4jC and 7.7 units/mL.

FDH

Enzyme activity was detected (Groger et al., 2004) with a

100 mM potassium phosphate buffer pH 7.5 containing

160 mM sodium formate and 0.25 mM NAD at 30jC and

340 nm (Mol ext. = 6.22 � mM�1cm�1). Candida boidinii

formate dehydrogenase [EC 1.2.1.2] (FDH) contained

70 units and 26.3 mg protein per mL. The CLEA was pre-

pared in saturated ammonium sulfate with 8 mM gluta-

raldehyde at 4jC and 7 units/mL.

Scale-Up and Preparative Use ofh-Galactosidase CLEA

Two hundred milligrams of h-galactosidase in 20 mL

potassium phosphate buffer pH 7.3 was added dropwise to

80 mL 2-propanol in a 250-mL flask with magnetic stirrer

immersed in a water bath at room temperature. After

complete addition 3.7 mL 25% glutaraldehyde was added

and the suspension was stirred for 1 h. Then, the CLEA was

centrifuged off, washed twice with 50 mL 50% ammonium

sulfate solution and subsequently stored in it. Either 4 mg of

free enzyme or an equivalent CLEA suspension (made from

4 mg) was added to 20 mL of a 50 mM lactose solution in

25 mM potassium phosphate buffer pH 7.3. After 3 h or

overnight incubation with magnetic stirring at room tem-

perature the reaction mixtures were centrifuged and the

supernatants concentrated in vacuo. 13C NMR showed 55%

conversion in both cases, while overnight incubation gave

quantitative hydrolysis. Upon assaying the recovered

CLEA, it was found that no activity was lost during lac-

tose hydrolysis.

Filtration

The filter system was supplied by CUNO Benelux. 0.2 to

5 A PPXL (polypropylene) filters and a 0.5 A Zeta Plus

050 HT filter were used. An amount of 100 mg CLEA was

suspended in 10 mL of demineralized water. The liquid

was pressed through the filter with 2 bar nitrogen gas. The

filtrate was then assayed for enzyme activity.

RESULTS AND DISCUSSION

Precipitation

To devise a high throughput method for optimization, the

preparation of CLEAs was divided into several steps (see

Scheme 1) consisting of (1) precipitant choice and precip-

itant concentration optimization followed by (2) optimizing

the cross-linker and protein concentration. First, the activity

recovery of the aggregated enzyme was determined, to

verify whether the precipitation step leaves the protein in

an active state before proceeding to cross-linking. For this

initial screening, precipitation was conducted with up to

90 vol % precipitant. The latter concentration leaves no

proteins in solution using the most polar precipitant.

756 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 87, NO. 6, SEPTEMBER 20, 2004

Downscaling was possible to only 10 AL (step 1), containing

a few units of activity, which turned out to be a reliable

volume for routine testing. Next, aggregation was induced

with 90 AL of (diluted) precipitant (step 2). At this stage the

presence of aggregates was not detected visually, although it

was indicated in some cases by the increased turbidity of the

suspension. Subsequently, the mixture was quenched with

900 AL buffer, giving complete resolvation of the protein

(step 3A). The remaining maximum of 9% precipitant is far

under the minimum amount necessary for precipitation.

Furthermore, it did not inactivate enzyme in any of the

examples. Aliquots were transferred onto microtiter plates

or into single cuvettes followed by the activity assay.

Comparison with initial activities indicated the effective-

ness of the aggregation step. The optimum precipitation

method was selected on the basis of this latter result. Using

microtiter plates instead of cuvettes allowed scanning in

one run for 12 different precipitants with 8 representa-

tive concentrations.

We found that quenching the enzyme solution with

high precipitant concentrations actually gave much better

results than—the up to now thought best—slow addition of

precipitant which is more analogous to crystallization. In

Table I it is shown that quenching of enzyme solutions with

90% precipitant yielded 100% activity for at least one of the

precipitants after resolvation. The reason for this high

retention of activity can be found in a different outcome of

the competition between aggregation and denaturation.

Quenching or even adding enzyme to precipitant results in a

shockwise aggregation in which time-interval there is little

chance for the protein to denature, although this latter

process can be very fast. The enzyme glucose oxidase

(Fig. 1) gave good activity yields with some precipitants at

90 vol %, however, with lower concentrations of precipitant

(< 70%) partial inactivation occurred because of incomplete

precipitation. The activity retained in the aggregated state

can, of course, only be determined after cross-linking. The

precipitation temperature had little effect. Even with the

enzymes showing poor activity recovery the difference in

recovery between room temperature and 4jC was mostly

negligible. Fluctuations in temperature are presumed to be

quickly dissipated by the vial because of the small volume.

Generally, mixing organic solvents with water will produce

heat, while ammonium sulfate chills the solution. However,

during scale-up excessive heat production can cause prob-

lems if it is not properly controlled. To determine the

amount of precipitant necessary to remove all activity from

the supernatant, samples must be centrifuged with sub-

sequent assaying of the supernatant.

Cross-Linking

The second stage of CLEA preparation is the cross-linking

of the aggregate (Scheme 1, step 3B). After the micro-

precipitation step, cross-linker is added. Alternatively, the

cross-linker can be added to the precipitant prior (Lopez-

Serrano et al., 2002) to the aggregation step; low con-

centrations of cross-linker would require very small

volumes to be transferred. After the required cross-linking

Scheme 1. High throughput set-up for CLEA optimization.

Table I. Activity of resolved aggregates using 90% (V/V) precipitant and activity after cross linking.

Precipitant (90%) CaLA CaLB TlL RmL GlcOx GalOx Laccase Gal-ase Trypsin ADH FDH Phytase Averagea

1 Buffer 100 100 100 100 100 100 100 100 100 100 100 100 100

2 Methanol 3 64 43 21 0 3 0 0 89 0 2 19 18

3 Ethanol 45 66 258 187 15 19 47 0 135 1 23 97 51

4 1-Propanol 48 30 1511 223 85 83 85 80 129 1 13 66 66

5 2-Propanol 43 77 169 95 104 100 99 82 144 6 55 93 79

6 tert-Butyl alcohol 142 100 1779 934 116 107 139 99 148 13 90 88 91

7 Acetone 107 52 178 706 94 87 58 65 185 24 95 77 79

8 Acetonitrile 100 75 561 428 116 93 27 88 151 21 84 79 81

9 DME 231 100 1013 561 113 88 78 79 142 7 50 95 83

10 Ethyl lactate 86 39 108 14 127 93 108 82 142 4 32 123 71

11 Sat. (NH4)2SO4 101 131 113 133 101 74 139 83 186 100 88 52 91

12 DMF 6 72 62 58 1 1 0 0 85 0 0 19 25

13 DMSO 5 107 95 43 2 0 0 0 131 0 0 5 29

14 PEG 115 138 141 102 114 52 186 100 153 44 81 80 88

after cross-linkingb 263 177 327 934 100 95 50 100 51 20 7 100 77

aMaximum contribution per enzyme = 100%.bFor precipitant, see Materials and Methods section.


time the now cross-linked aggregates are quenched with

buffer (step 4). A sample is withdrawn from the resulting

suspension, which contains CLEA as well as residual free

enzyme, and assayed for activity. Then, the CLEA is cen-

trifuged off and again a sample is withdrawn from the

supernatant (step 5), which now contains only free enzyme.

The difference in activity between step 4 and step 5 is the

CLEA activity (see Fig. 2). This approach is the most simple

and accurate way of determining the activity of the CLEA,

without having to wash and redisperse, which will increase

clotting and thereby mass-transport limitations (see Proper-

ties of CLEAs section below). In principle, the precipitant

with the highest recovery of activity is the best starting point

for cross-linking. However, aggregate activity might differ

from the redissolved enzyme (step 3A), making it necessary

to use multiple precipitants and assay the activity of the

resulting CLEAs. In addition, the cross-link time is of

indirect influence since this sets the exposure time of the

enzyme to the precipitant.

Although various cross-linkers are known and can be

used, glutaraldehyde remains a cheap and very versatile

agent. An in-depth study of cross-linkers particularly suit-

able for preparing CLEAs is the subject of another inves-

tigation currently conducted in our group. Because some

enzymes are inactivated by glutaraldehyde, optimization

often means minimizing the amount of cross-linker. In the

case of CaLB, higher glutaraldehyde concentrations (up to

150 mM) lead to hyperactivation. On the other hand, alcohol

dehydrogenase was completely inactivated when the glu-

taraldehyde concentration exceeded 10 mM (see Fig. 3); a

concentration above which the hyperactivation of CaLB just

began to emerge, surprisingly. The time needed for com-

plete cross-linking depends on the temperature. Generally,

at room temperature no increase in cross-linking was ob-

served after 3 h. Leaching was never observed, although a

CLEA might contain some uncross-linked aggregated en-

zyme. Simple washing, e.g., overnight incubation in buffer

will remove most free enzyme.

After selecting the best precipitant the influence of the

protein concentration was maximized as well. This is

desirable to maximize the amount of CLEA produced per

unit volume. The presence of salts in the enzyme solution

manifests itself in the aggregate sticking to the vial making

it necessary in those cases to dilute the enzyme with

water prior to precipitation. For CaLB (see Fig. 4) hyper-

activation reached a tentative maximum with a medium

amount of protein, clearly demonstrating the need for high-

throughput optimization.

Scale-Up and Preparative Use

Dropwise addition of an aqueous enzyme solution to the

precipitant made the scaling-up of CLEA preparation easier

and without unexpected activity loss. The aggregation time

is the same for each drop added and the aggregates are stable

in the precipitants during this interval. To establish the

validity of the parameters found in the high-throughput

precipitation the enzyme h-galactosidase was subjected to

a thousand-fold scale-up: from 0.1 mL to 100 mL. The ac-

tivity recovery was exactly the same in both cases demon-

strating both accuracy and viability of the parameters found

with this method. Hydrolysis of lactose mediated by free

enzyme vs. the CLEA, monitored by NMR, showed equal

chemical yields. After completion of the reaction the CLEA

was centrifuged off, washed and assayed for activity,

showing no significant loss of activity. Obviously, the

Figure 1. Precipitation of glucose oxidase. The activity shown is

measured after resolvation of the aggregate in buffer.

Figure 2. Pellet and supernatant activity during cross-linking of

Candida antarctica lipase B aggregate with increasing glutaraldehyde

concentrations.

Figure 3. Optimized CLEA activities of lipase B from Candida an-

tarctica (CaLB) and alcohol dehydrogenase from Rhodococcus erythropo-

lis (ADH).


mechanical stability, influenced by detrimental operations

such as high stirring rate or the bubbling of oxygen is

excellent when using CLEAs.

Structure of CLEAs

One major property of CLEAs has been—up to now—

relatively unexplored: their particle shape and size. The

number of enzyme molecules and the way they are packed

together in an aggregate can be expected to have a crucial

influence on the activity of the aggregate as a whole. Hence,

knowing the factors of influence and being able to control

them would pave the way for changing the CLEA from a

phenomenon into a mature, well-defined catalytic particle.

During aggregation the solubility of the enzyme in the

surrounding medium decreases. When this process is slow

the enzyme might denature because of the severe force

exerted on its structure. If it is able to find neighboring

protein molecules in time to surround it, chances are fairly

good that it will retain its tertiary structure. As seen from the

high-throughput experiments, increasing the speed of ag-

gregation in cases of poor activity recovery, always gave a

completely active aggregate. Now the question is, to what

extent will enzymes aggregate? Most likely, the surface

tension of the aggregate will set the diameter. It is similar to

water/oil mixtures where small droplets (emulsion) are

formed. Cell- or droplet-like structures of protein aggre-

gates, depending on the surface hydrophobicity, can in that

case be expected. The interplay between free energy in-

crease by interfacial surface formation and free energy de-

crease in solids formation governs the critical nucleus size

according to the classical nucleation theory. The final

aggregate size of the primary particles is then governed by

the ratio of nucleation and growth, but will likely be small.

Scanning electron microscopy showed a very uniform

structure of the aggregates. From the different enzyme ag-

gregates we examined, two types emerged. Type 1 aggre-

gate was formed by Candida antarctica lipase B (Fig. 5).

This enzyme is scarcely glycosylated and highly lypophilic.

The diameter of the aggregate is about 1 A with a small

deviation. Taking an enzyme size for CaLB of 5 � 5 �5 nm, a single CLEA particle contains a maximum 8 � 106

enzyme molecules. Aggregates built from enzymes with a

more glycosylated surface or a surface with more hydro-

philic residues were found to be smaller, approximately

0.1 A in diameter, called Type 2 aggregates. Candida rugosa

lipase (Fig. 6) and Prunus amygdalus R-oxynitrilase are

examples of that type. These enzymes are glycosylated and

therefore have a more hydrophilic surface. One CLEA

particle of this aggregate contains a maximum of 8 � 103

enzyme molecules. From Figures 3 and 4 it is apparent that

enzyme activity in these ‘‘balls’’ is not only fully retained;

they can be in a hyperactivated state reflected by activities

exceeding 100%. Although Type 1 aggregates can contain a

thousand times more enzyme molecules than Type 2, in both

cases the enzymes find themselves in an apparently ideal

environment where their natural function is thriving. Since

the enzyme molecules are packed together in a small

volume compared to the free protein in solution, one might

expect mass-transport limitations, particularly with fast

reactions. If the CLEAs are finely dispersed in solution—as

is the case when the cross-link step is completed and sub-

sequently quenched—this effect was actually small.

Figure 5. Candida antarctica lipase A/B CLEA, 1 CLEA particle can

contain up to 8 million enzyme molecules (magnification 3500�).

Figure 6. Candida rugosa lipase forms smaller CLEAs. One CLEA

particle can contain up to 8000 enzyme molecules (magnification

25000�).

Figure 4. Effect of the volumetric activity on CLEA activity of Candida

antarctica lipase B. The dashed line represents units CLEA/free = 1.


Clustering of Aggregates

While Type 1 aggregate forms typical ‘‘balls,’’ Type 2 ag-

gregate clusters into less-defined structures. CLEAs can

form larger clusters (Figs. 7 and 8), which do have mass-

transport limitations, especially in fast UV tests. The size of

these clusters can be up to 100 A, making them visible to the

naked eye. The number of CLEAs in a cluster is much less

uniform than enzymes in an aggregate: it can vary from a

few to a few hundred thousand (Fig. 8 and Table II). The

previous findings with laser scattering (data not shown) that

suggested a variable number of enzyme molecules per ag-

gregate is now rationalized as being a variable number of

aggregates per cluster.

Centrifugation leads to increased cluster formation.

When a dispersed CLEA is assayed for activity, directly

after dilution of the cross-link medium, a higher activity is

observed than when the sample is centrifuged and redis-

persed. This treatment presumably does not disturb the

individual CLEA and thereby the enzyme structure (as is

obvious from the SEM analysis), but it does squeeze the

CLEA particles close together elevating mass-transport lim-

itations to a noticeable level. The interaction between par-

ticles can be either reversible, as is demonstrated when these

clusters are put in aqueous organic solvents—which causes

them to break up—or irreversible when active cross-link

residues on the CLEA surface form covalent bonds between

individual particles. Some differences between enzymes

were observed: with CaLB very large and hydrophobic

clusters were obtained whereas with h-galactosidase well-

dispersible suspensions were common. The most noticeable

structural difference between these two enzymes is that

h-galactosidase is extensively glycosylated and CaLB is

not. Comparing activities for CaLB CLEA, found in the

relatively fast hydrolysis of p-nitrophenyl propionate mea-

sured via UV/Vis absorption, with the slower hydrolysis of

triacetin (monitored by titration) the mass-transport limi-

tation was obvious. Compared with free enzyme the

first showed 35% activity recovery and the latter 177%.

For h-galactosidase however, activity recovery found in

the hydrolysis of p-nitrophenyl-h-d-galactopyranoside and

in lactose hydrolysis was the same. These two CLEAs em-

phasise the important effect of cluster formation on the

apparent activity.

Isolation of CLEAs

One major advantage of CLEAs is their facile separation

from aqueous solutions. In contrast to free enzyme, brief

centrifugation results in complete recovery of the catalyst.

Another way of isolating CLEAs from a reaction mixture is

filtration. The aggregate or cluster size determines how

Figure 7. Candida antarctica lipase AB CLEA clusters in water (magnification 150�).

Figure 8. Particle size analysis of Candida antarctica lipase AB CLEAs.

Clusters can contain more than 100,000 single aggregates.


successful filtration will be. Certainly, the large clusters do

not pose any problem for even a large-pore glass filter and

the single submicrometer aggregates can be filtered with

low-molecular-weight membrane filters. For medium-size

clusters the widely used micrometer pore-size filters can be

used. We applied several filters ranging from 0.2–5 A. As

expected, the 1 A size Candida antarctica lipase CLEAs

passed through filters with pore sizes ranging from 1–5 A.

More surprisingly, they also passed through the 0.2 Afilters demonstrating the elasticity of the ‘‘enzyme balls.’’

This suggests that the individual enzymes in an aggregate

do have some freedom of movement that is undoubtedly

beneficial for their activity. Smaller aggregates like the ones

formed from Candida rugosa lipase elegantly showed the

effect of clustering. With their 0.1 Am size they could be

filtered with 0.5 A filters. Although the polypropylene

(PPXL) filters were completely blocked, filtration was pos-

sible with the Zeta Plus filters, which have a graded density

resulting in better cake building. In Figure 6 it can be seen

that the space between the CLEAs is filled up, presumably

with protein, as the weight and volume contribution of the

cross-linker is negligible. Controlling the size of the clusters

means controlling the ability to filter the CLEAs.

CONCLUSIONS AND OUTLOOK

We have shown that by a suitable optimization of the pro-

cedure, which may differ from one enzyme to another, the

CLEA methodology is applicable to essentially any enzyme

including crude preparations, affording stable, recyclable

catalysts with high retention of activity. The method is

exquisitely simple and amenable to rapid optimization. The

mechanical stability, influenced by typically detrimental

operations such as high stirring rate or the bubbling of

oxygen necessary for many oxidases, poses little problems.

Furthermore, no inactivation by foam formation is observed

when using CLEAs.

We have established the optimization of crucial factors

such as precipitant, precipitant concentration, cross-linker

concentration, and protein concentration. Any parameter

influencing enzyme activity can be optimized quickly with

the presented method. In principle, the approach should be

iterative, however, taking optimum conditions rapidly from

one test to the next to afford a quantitative activity yield.

Moreover, the optimum conditions found were completely

representative for even a 1000-fold scale-up, giving the

scaled down CLEA optimization method extra appeal, since

many conditions can be tested with small amounts of en-

zyme. In contrast to CLECs, there is no need for the enzyme

to be crystallized and the technique can be applicable to the

preparation of combi CLEAs containing two or more

enzymes. The spherical structure of the CLEAs offers new

insights in CLEA behavior. Future research is focusing on

designing CLEA properties by applying different agents for

pre- or postaggregation modification of the enzyme as well

as the CLEA, particularly those influencing clustering be-

havior. Control over clustering would finally turn CLEAs

into well-defined catalytic particles.

The authors wish to thank Gerard de Vos from the Polymer Materials

and Engineering Group, Delft University of Technology for the

scanning electron microscopy and Stef van Hateren from the Bio-

separation Technology Group for performing light microscopy.

Thanks are due to DSM (Delft, The Netherlands, h-galactosidase and

phytase), Julich Fine Chemicals (Julich, Germany, laccase, alcohol

dehydrogenase, and formate dehydrogenase), Novozymes (Bags-

vaerd, Denmark, lipases and trypsin), Hercules (Barneveld, The

Netherlands, galactose oxidase), CLEA Technologies (Delft, The

Netherlands, oxynitrilase CLEAs) for generous gifts of enzyme, and

CUNO Benelux (Zwyndrecht, The Netherlands) for the filter system.

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2 5.3 2.7 2.1 11

3 4.4 2.7 1.6 7

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5 1.7 1.6 1.0 2

6 1.7 1.6 1.0 2

7 3.8 3.2 1.0 4

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9 109 15.7 9.7 1,057

10 1.1 1.0 0.5 1

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preparation, optimization, and structures of cross-linked enzyme aggregates (cleas)

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