1256 Catal. Sci. Technol., 2011, 1, 1256–1261 This journal is c The Royal Society of Chemistry 2011

Cite this: Catal. Sci. Technol., 2011, 1, 1256–1261

Poly-lysine supported cross-linked enzyme aggregates with efficient

enzymatic activity and high operational stabilityw

Hiroshi Yamaguchi,za Masaya Miyazaki,*ab

Yuya Asanomiaand Hideaki Maeda

ab

Received 10th March 2011, Accepted 19th July 2011

DOI: 10.1039/c1cy00084e

In this study, the operational stability of enzymes in a cross-linked aggregate (CLEA) formed

with poly-lysine was examined. Chymotrypsin, subtilisin, citrate synthase and laccase, which

are structurally and mechanistically diverse, were used as model enzymes. The preparation of

poly-lysine supported CLEA was completed within 3 h. The immobilized enzymes were more

stable than free enzymes at high temperature, in the presence of a chemical denaturant or

in an organic solvent and were recycled without appreciable loss of activity. In addition, the

immobilized proteases showed higher or similar hydrolytic activity in acidic pH than in neutral

pH. This immobilization method was also applicable to the multi-subunit protein. These results

suggest that the poly-Lys supported CLEA can be used as catalysts with own enzymatic activity

and high operational stability.

Introduction

Enzymes have high catalytic activity and substrate specificity.

Consequently, they are often applied as catalysts in industrial

synthesis.1–3 However, because they lack operational stability

and reusability, an important limitation hinders the use of

enzymes in wider applications. Enzyme immobilization

presents one means to overcome these difficulties.4,5 Immobilized

enzymes present several advantages. An important benefit is

their often-enhanced stability against thermal or chemical

denaturation and autolysis (when a protease is immobilized),

which provides high operational stability and reusability.5

Furthermore, the immobilized enzyme can be isolated from

the product easily, requiring no purification steps. Because of

these interesting features, many applications using the

immobilized enzymes have been reported recently, including

organic synthesis,4,5 proteolysis in proteomic analysis,6–8

biosensors and protein microarrays.9

Enzyme immobilization is usually conducted using a

non-covalent binding method or a covalent binding method.9

The non-covalent binding method is often performed using an

affinity interaction such as a histidine-tagged enzyme to a

chelating-agarose gel.4,9 Although the binding are specific

interactions, it can be used only for the recombinant tagged

enzymes. In contrast, the covalent binding methods are

achieved by chemical reactions between the side-chains in

protein and a solid support such as modified glass or poly-

methylmethacrylate. These methods require no modification

of the target enzymes. Therefore, they are widely applicable.

However, it is difficult to control the immobilization yield

because multiple reaction points exist in these covalent binding

methods. Furthermore, the enzyme’s conformation is often

altered, engendering reduction of its catalytic activity and

operational stability.10,11 In addition, the reported procedures

for producing immobilized enzymes include multi-step

procedures necessitating considerable time and effort.7,12,13

Therefore, a facile preparation procedure of the immobilized

enzymes is sought for routine use.

Cross-linked enzyme aggregation (CLEA) has been

reported as a carrier-free immobilization method.5,14 Generally,

CLEA is prepared by precipitating the enzyme with addition

of a salt or an organic solvent with a subsequent cross-linking

reaction by a cross-linker such as glutaraldehyde (GA). How-

ever, the cross-linking reaction might not be as effective

as expected for enzymes with low Lys residue contents.

Reportedly, the addition of albumin, which forms co-aggregates

with the enzyme with a low Lys residue content, can form CLEA

with activity.15,16 Nevertheless, this method was often insufficient

to improve the stability.16 Consequently, this is not a universal

method. We previously reported a method by which the enzyme

was immobilized on the inner wall of the microchannel through a

cross-linking polymerization between Lys residues on the mixture

of acidic enzyme and poly-lysine (poly-Lys).17 Acylase, which

aMeasurement Solution Research Center, National Institute ofAdvanced Industrial Science and Technology, Tosu, Saga 841-0052,Japan. E-mail: m.miyazaki@aist.go.jp; Fax: +81-942-81-3627;Tel: +81-942-81-4059

bDepartment of Molecular and Material Sciences,Interdisciplinary Graduate School of Engineering Science,Kyushu University, Kasuga, Fukuoka 816-8580, Japan

w Electronic supplementary information (ESI) available: Results ofpoly-Lys on CLEA formation and hydrolytic activity for CT-CLEAand SBc-CLEA. See DOI: 10.1039/c1cy00084ez Present address: Liberal Arts Education Center, Aso Campus, TokaiUniversity, Aso, Minamiaso, Kumamoto 869-1404, Japan.

CatalysisScience & Technology

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has an isoelectric point (pI) of 4.0, was immobilized using this

method, and the obtained acylase-CLEA showed pH stability.17

Moreover, protease-CLEA prepared using this method

showed rapid proteolysis and high stability against tempera-

ture, urea, and organic solvents.8,18 From these results, we

infer that poly-Lys supported enzyme immobilization is

applicable not only for acylase and proteases but also for

other enzymes, and that the prepared CLEAs can be useful for

catalysts with high operational stability. To examine this

hypothesis, the poly-Lys supported CLEA formation was

applied in this study for four enzymes, which are structurally

and mechanistically diverse. Their enzymatic activity and

operational stability were studied.

Experimental

Materials

Chymotrypsin (CT, from bovine pancreas), GA and para-

formaldehyde (PA) were obtained from Wako Pure Chemical

(Osaka, Japan). Citrate synthase (CS, from porcine heart) in

an ammonium sulfate suspension, acetyl-CoA, 5,50-dithiobis

nitrobenzoic acid, N-glutaryl-Phe-p-nitroanilide (GPNA),

laccase (Lac, from Trametes versicolor), oxaloacetic acid,

poly-L-Lys hydrobromides (70400 Da, 32200 Da and 4200 Da),

subtilisin Carlsberg (SBc, from Bacillus licheniformis) and

N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (Suc-AAPF-pNA)

were purchased from Sigma-Aldrich (St. Louis, MO, USA). In

addition, 2,20-azino-di-(3-ethylbenzothiazoline-6-sulfonate)

(ABTS) was purchased from KPL (Gaithersburg, MD, USA).

All other chemicals were of analytical grade.

Formation of poly-Lys supported CLEAs and a

turbidity measurement

Proteases (CT and SBc) and poly-Lys were dissolved in 50 mM

phosphate buffer (PB, pH 8.0) at concentrations of 10 mg mL�1

and 20 mg mL�1, respectively. The cross-linker reagent

containing GA (0.25%, v/v) and PA (4%, v/v) in 50 mM PB

(pH 8.0) was used at a ratio of 1 : 16 (v/v).17 All solutions were

filtered using a 0.45 mm polypropylene cellulose syringe filter

(Minisart RC4; Sartorius Stedim Biotech, Goettingen,

Germany) before use. The enzyme, poly-Lys and the cross-

linker were mixed in a 96-well plate (Nunc, Kamstrup, Denmark).

After different incubation times at 4 1C, the turbidity of themixture

was measured by the absorbance at 630 nm using a spectro-

photometer (Multiskan JX; Thermo, Waltham, MA, USA).

Preparation of poly-Lys supported CLEAs for an enzymatic

activity measurement

The CS solution was prepared using dialysis against 50 mM

PB (pH 8.0) to remove ammonium sulfate before use. Other

enzymes were dissolved in 50 mM PB (pH 8.0) without

dialysis. Concentrations of proteins were determined using

the Coomassie Plus Bradford assay (Pierce, Rockford, IL,

USA). The stock concentrations of proteases (CT and SBc),

CS and Lac were 10 mg mL�1, 634 mg mL�1 and 969 mg mL�1,

respectively. The enzyme, poly-Lys and the cross-linker were

mixed at a volume ratio (40 mL : 40 mL : 80 mL) in a test-tube

and incubated at 4 1C for 2 h. Then CLEA was collected using

centrifugation (12 000 rpm for 10 min). It was rinsed with

200 mL of 1 M Tris–HCl (pH 8.0) for 20 min at 4 1C, which

simultaneously quenched any active aldehyde group. To

reduce the resulting Schiff base, CLEA was treated with

200 mL of 50 mM NaCNBH3 in 50 mM borate buffer

(pH 9.0) for 20 min at 4 1C. Then it was washed with 20 mM

PB (pH 7.5). The immobilized enzyme in the CLEA matrix was

analyzed using the BCA protein assay (Pierce) or absorbance at

280 nm using an uncross-linking enzyme fraction.

CT and SBc hydrolytic activity measurements

Typical measurements of hydrolytic activity using CT-CLEA

and SBc-CLEA were conducted in 20 mM PB (pH 7.5) at

30 1C for 20 min (CT) or 10 min (SBc). The GPNA for CT

(1 mM) and Suc-AAPF-pNA for SBc (200 mM) were used as

substrates. The chemical stability of CLEA was tested in a

reaction buffer with urea (1 to 7 M) at 30 1C. The thermal

stability of CLEA was tested in 50 mM PB (pH 7.5) at 30 or

50 1C. For pH stability, CLEA was incubated in buffer of

several pH values (5.5–9.0) at 30 1C, using acetate buffer for

pH 5.5, PB for pH of 6.0–8.0 or borate buffer for pH 9.0. The

kinetic parameters, Km and kcat, were measured at various

substrate concentrations. The data were fitted to the Michaelis–

Menten equation.18 The reaction was evaluated as the amount

of released p-nitroaniline measured from absorbance at

405 nm. For in-solution experiments, the concentrations of

free CT and free SBc were 50 and 2 mg mL�1, respectively.

Lac enzymatic activity measurement

The activity was typically measured at 30 1C for 10 min in

100 mM acetate buffer (pH 4.5), for which ABTS (50 mM) was

used as a substrate. For pH stability, CLEA was incubated in

buffer with several pH values (4.5–9.0). Other reaction condi-

tions are described above. The absorbance at 405 nm was

measured at room temperature using a spectrophotometer.

For in-solution experiments, the free Lac concentration was

5 mg mL�1; hydrochloric acid (1 M) was added to the solution

to stop the reaction.

CS enzymatic activity measurement

CS-CLEA was added to a reaction mixture containing 20 mM

Tris–HCl (pH 7.5), 300 mM acetyl-CoA, 100 mM oxaloacetic

acid and 100 mM 5,50-dithiobis nitrobenzoic acid.19,20 The

reaction was conducted with or without 3 M urea at 30 or

50 1C for 20 min. For in-solution experiments, the free CS

concentration was 1 mg mL�1.

Results and discussion

Poly-Lys supported enzyme immobilization

A typical procedure for cross-linking an enzyme involves

activation of the primary amine groups of the enzyme with a

cross-linker to create aldehyde groups that can react readily

with other primary amine groups of enzymes.4,7,21 Cross-

linking yields depend on the number of the Lys residues of

the enzyme. Therefore, the acidic or neutral enzyme (pIo 7.0)

cannot be cross-linked efficiently merely by the use of a

cross-linker. Although high concentration of a cross-linker

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can increase the cross-linking yield, it often causes a change

of its conformation, engendering a reduction of its catalytic

activity.10,11 To overcome this difficulty, poly-Lys was used as

the amine donor in this study. It is expected that the large

number of primary amine groups of poly-Lys can improve the

cross-linking yield with lower concentration of cross-linker than

those in reported procedures (typically 5–10% of GA, v/v).22,23

Based on a similar idea, pentaethylenehexamine and albumin

were used to cross-link the chloroperoxidase enzyme (pI= 4.1).15

The preparation steps of CLEA are presented in Fig. 1. As a

cross-linker, the mixture of GA and PA was used at a ratio of

1 : 16 (v/v).21 The resulting Schiff base was reduced by

NaCNBH3. To test the usability of our method for several

types of enzymes, we prepared CLEAs using CT, SBc, Lac and

CS. Their pI values were 8.4, 6.6, 5.8 and 7.0, respectively.

Although the pI value of CT is close to the pH value of the

reaction buffer (8.0), the reaction without poly-Lys showed

low cross-linking yield (data not shown). The other three

enzymes were also not efficiently cross-linked without poly-

Lys under our experimental conditions, suggesting that only

Lys residues on the surface of the enzymes are insufficient for

cross-linking.

Effect of poly-Lys concentration on CLEA formation

To optimize the cross-linking reaction, we first studied the

ratio of poly-Lys and enzyme. The CT and SBc that were

frequently applied to study the enzymatic reactions for organic

synthesis24,25 were used. The molecular weight of poly-Lys was

70 kDa. Different concentrations of poly-Lys (1.25, 2.5 and

5.0 mg mL�1) were mixed with CT or SBc at constant concen-

tration (2.5 mg mL�1). The mixture was cross-linked by GA

(0.25%, v/v) and PA (5%, v/v) in 50 mM PB (pH 8.0). To avoid

autolysis of protease in bulk solution during the cross-linking

reaction, the preparation of CLEA was conducted at 4 1C.18

The CLEA formation was evaluated according to the

reaction mixture turbidity.17 The increase in poly-Lys

concentration increased the turbidity (Fig. S1w), suggesting

that enzymes were efficiently cross-linked in the presence of

poly-Lys. One- or two-fold poly-Lys concentration was suffi-

cient to form CLEA. After reaction for 2 h, more than 75% of

CT and more than 65% of SBc had been cross-linked. No

turbidity was observed when mixing the enzyme and poly-Lys

without a cross-linker.

Both CLEAs showed their own hydrolytic activity (Fig. S1,

ESIw), indicating that both proteases maintained their active

conformations in the CLEA matrices. CT-CLEAs prepared

using equivalent poly-Lys concentration with CT concentra-

tion showed higher activity than that by low poly-Lys

concentration (Fig. S1A, ESIw). Leakage of free CT from

the CLEA matrix was not observed during washing steps

when high poly-Lys concentration was used. In contrast,

SBc-CLEAs showed similar immobilization yield (Z 65%)

and activity (490%) in all cases (Fig. S1B, ESIw). These

results suggest that acidic SBc (pI = 6.6) can interact easily

with basic poly-Lys, even at low poly-Lys concentration, while

basic CT (pI = 8.4) requires high concentration of poly-Lys.

This suggestion is supported by the results obtained for Lac,

CS (see below) and alkaline phosphatase (pI = 5.9), which

need no great amount of poly-Lys for immobilization.8

The molecular weight (MW) of poly-Lys can also affect the

interaction between the enzyme and poly-Lys. Therefore, we

studied the formation of CT-CLEA using different MWs of

poly-Lys (32 kDa and 4 kDa). The CT and poly-Lys were mixed

at a ratio of 1 : 1 and the cross-linking reaction was performed

for 2 h at 4 1C. The turbidity measurement revealed that the

advantage of lower MWs (32 kDa and 4 kDa) was lower CLEA

formation than that inMW of 70 kDa (data not shown). Further

experiments were performed using poly-Lys with 70 kDa.

Effect of cross-linker concentration on CLEA formation

We next optimized the cross-linker concentration for prepara-

tions of CT-CLEA and SBc-CLEA. Based on the above

results, protease and poly-Lys were mixed at a ratio of 1 : 1,

and the cross-linking reaction was performed for 2 h at 4 1C.

Using various amounts of the cross-linker mixture, we

found that optimal concentrations of PA and GA for

CT-CLEA were 2% (v/v) and 0.125% (v/v), respectively

(Fig. S2Aw). The activity recovery was 45% at 30 1C. Further

increasing the cross-linker concentration produced similar

immobilization yields (ca. 75%) and did not change the

activity (ca. 40%). In contrast, CT-CLEA prepared with a

low concentration of cross-linker (PA= 0.5%, v/v and GA=

0.031%, v/v) showed much lower immobilization yield and

activity than those of other CT-CLEAs. For SBc-CLEA, the

concentration of the cross-linker did not affect the immobili-

zation yield (ca. 60%) or activity (Z 90%) at 30 1C (Fig. S2B,

ESIw). Moreover, no leakage of free SBc was observed using a

low concentration of cross-linker. These results suggest that

the acidic enzymes were sufficiently immobilized at low

concentrations of cross-linker in the presence of poly-Lys.

Thermal stabilities of protease-CLEAs

We studied thermal stabilities of CT-CLEA and SBc-CLEA. The

activities of both CLEAs at 50 1C are depicted in Fig. S2 (ESIw).

Fig. 1 Preparation of the poly-Lys supported CLEA. The cross-

linking reaction between the aldehyde groups of a cross-linker and

amino groups present in the enzyme and/or poly-Lys forms reversible

imine groups (Schiff base). To form stable amine groups, the CLEA

matrix was reduced by NaCNBH3.

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At 50 1C, free CT showed lower activity than that at 30 1C

(data not shown). In contrast, CT-CLEAs at 50 1C showed

1.6-fold higher activity than those at 301C (Fig. S2A, ESIw),indicating that the immobilized CT has thermal stability and

that diffusion of the substrate moving into the CLEA matrix

was increased at 50 1C.

Reportedly, subtilisin from Bacillus subtilis showed high

thermal stability.26 As reported, free SBc were not denatured

at 50 1C and showed hydrolytic activity (data not shown). All

five SBc-CLEAs kept at 30 1C retained their activity (490%

in Fig. S2B, ESIw), indicating that the immobilized SBc in the

CLEA matrix also maintained thermal stability.

pH profiles of protease-CLEAs

Immobilized enzymes often produce a shift in the optimum

pH.15,17,26 Therefore, the effects of pH on the activity of

CT-CLEA and SBc-CLEA were examined at pH 5.5–9.0 at

30 1C (Fig. 2). Free CT and free SBc are well known to show

maximum hydrolytic activity at around pH 7.0. In contrast,

the pH profiles of both protease-CLEAs were broader than

those of free proteases. An interesting difference is that both

protease-CLEAs maintain their hydrolytic activity, even at

pH 5.5. Because the enzymatic reaction at broad pH can be

useful for catalysis in chemical and biotechnology fields,

kinetic parameters by CT-CLEA and SBc-CLEA at pH 7.5

and pH 5.5 were measured (Tables S1 and S2w). The

CT-CLEAs prepared using 2–3% (v/v) of PA and

0.125–0.187% (v/v) of GA showed higher kcat/Km than other

CT-CLEAs exhibited at both pH conditions (Table S1, ESIw).At pH 7.5, kcat/Km values of CT-CLEAs were slightly less

than those of free CT, probably because of the diffusional

resistance of the substrate moving into the CLEA matrices.

In contrast, at pH 5.5, kcat/Km values of CT-CLEAs were

5.6-fold higher than those of free CT. Moreover, Km values of

CT-CLEAs were 7-fold lower than those of free CT at pH 5.5.

A lower Km value is known to represent higher binding affinity

between the enzyme and substrate, therefore suggesting that

basic CLEA matrices with high poly-Lys contents probably

interact easily with an acidic substrate (GPNA) at pH 5.5 than

at pH 7.5. Because all SBc-CLEAs prepared using different

cross-linker concentrations showed similar hydrolytic activity

(Fig. S2B and 2B, ESIw), two SBc-CLEAs that were prepared

using lowest and highest cross-linker concentrations were

tested (Table S2, ESIw). At pH 7.5, kcat/Km values of both

SBc-CLEAs were approximately 400-fold less than that of the

free enzyme, probably because of the mass transfer limitation

of the substrate in the SBc-CLEA matrix. In contrast, the

kcat/Km value of free SBc at pH 5.5 was 5.8-fold lower than

that at pH 7.5. Nevertheless, SBc-CLEAs maintained their

kcat/Km values. These results suggest that both proteases in

the CLEA matrices maintained their active conformations,

leading to their own activities at pH 5.5.

Hydrolysis activity of protease-CLEAs in urea or DMSO

solution

We next studied the chemical stability of the immobilized

enzyme against urea at 30 1C. Estimated [urea]1/2 of CT-CLEA

(PA of 2%, v/v and GA of 0.125%, v/v), which is the necessary

concentration of urea for half of the hydrolytic activity at 30 1C,

was 6.6 M, whereas that of free CT was 3.4 M. It is indicated

that the immobilization of CT increased the chemical stability.

In contrast, free SBc was not denatured, even at 7 M urea.

Therefore, we did not test the stability of SBc-CLEA.

The reaction in the organic solvent is important for the

synthetic application of catalyst. Therefore, the hydrolytic

reactions by protease-CLEAs (PA of 2%, v/v and GA of

0.125%, v/v) were performed in several organic solvents.

Protease needs molecular water for its hydrolytic reaction.

Therefore, DMSO, which is a miscible solvent with water, was

used. The residual activities of CT-CLEA and SBc-CLEA in

40% (v/v) DMSO were 27% and 59%, respectively, although

free proteases showed lower activity (o20%). The decrease in

activity in DMSO might result from the stripping of water

from the enzyme surface.26 It is possible that the CLEA matrix

can retain water even in organic solvent. Therefore, the

immobilized proteases were possibly more stabilized than free

proteases. Similar stabilities of protease-CLEAs were observed

in ethanol, 2-propanol and THF (data not shown).

Fig. 2 pH profile of CT-CLEA (A) and SBc-CLEA (B) prepared

using different concentrations of PA (%, v/v) and GA (%, v/v).

Hydrolytic reactions were conducted in 20 mM PB (pH 7.5) at

30 1C. Substrate: GPNA (1 mM) for CT-CLEA; Suc-AAPF-pNA

(200 mM) for SBc-CLEA. Concentrations of free CT and free SBc were

50 and 2 mg mL�1, respectively. The graph shows the mean � standard

error for at least three experiments.

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Stability of Lac-CLEA

Lac, an industrially useful polyphenol oxidase, has four

copper atoms in its catalytic site. Recently, Lac members have

been applied for organic synthesis27 or degradation of recalci-

trant compounds.28,29

Based on the results described above, Lac and poly-Lys

were mixed at a ratio of 1 : 1. Then the cross-linking reaction

was performed for 2 h at 4 1C. The pH profiles of Lac-CLEAs

that had been prepared using different cross-linker concentra-

tions are presented in Fig. 3. As free Lac, Lac-CLEAs showed

the maximum activity, with the optimum pH of around 4.0.

However, the activity recovery of Lac-CLEAs was lower than

that of free Lac. Moreover, the stabilities of Lac-CLEAs

against urea and DMSO were not improved (data not shown).

In earlier papers, when Lac was immobilized using GA, Schiff

base in the cross-linked Lac was not reduced by reducing

reagents.16,29 Although those reports provided no description

or explanation of reduction steps, the copper atoms in the

catalytic site were possibly affected. These data imply that our

poly-Lys supported enzyme immobilization is inapplicable to

the enzyme with metal atom(s) in its catalytic site.

Stability of CS-CLEA

CS synthesizes citrate from oxaloacetic acid and acetyl-CoA.

It is recognized to be of low operational stability.19,20 In

addition, to express its activity, CS must form a dimer

comprising two identical subunits. Therefore, it is expected

that CS is a good model enzyme to test not only the opera-

tional stability, it is also useful as an application of our

immobilization procedure for a multi-subunit enzyme.

For 2 h at 4 1C, CS, two-fold poly-Lys and the cross-linker

(GA of 0.25%, v/v and PA of 4%, v/v) were mixed. Under

these conditions, free CS was cross-linked almost completely

in CS-CLEA. As depicted in Fig. 4, CS-CLEA showed its own

activity, indicating that CS formed a dimer in the CLEA

matrix. The CS-CLEA stability against thermal denaturation

and urea denaturation was tested. Because the activity measure-

ment using 5,50-dithiobis nitrobenzoic acid19 is conducted for a

neutral pH solution, the assay was carried out at pH 7.5.

At 3 M of urea, CS-CLEA and free CS showed 66% and 9%

activity, respectively. The estimated [urea]1/2 of free CS at

30 1C was 1.1 M. Therefore, CS-CLEA was more stable than

free CS. In addition, CS-CLEA showed higher activity at

50 1C than free-CS did. It is suggested that, because of multi-

point cross-linking between CS and poly-Lys, the immobilized

CS increased stability although free enzyme was thermally or

chemically denatured. These data show that the poly-Lys

supported CLEA can be useful for immobilization of the

multi-subunit protein.

Reusability

The reuse of the immobilized enzyme as a catalyst is important

for a routine enzymatic reaction. Therefore, the reusability of

CLEAs was tested. After each reaction cycle, CLEAs were

washed with buffer solution and stored at 4 1C for more than

3 months. During this period, CLEAs were used for over 30

reaction cycles for the stability experiments described above.

The respective residual activities of CT-, SBc- and CS-CLEAs,

even after experiments of urea denaturation or thermal

denaturation, were almost identical to those obtained before

enzymatic reactions (490%), indicating that these CLEAs

maintained their activity after many uses. Additionally, no free

enzymes were released from CT-, SBc- and CS-CLEAs, as

measured using UV absorbance at 280 nm. Regarding free

enzymes, they lost their activity almost completely within five

days because free CS and Lac had formed precipitates and free

proteases (CT and SBc) were digested by autolysis. Although

the residual activity by Lac-CLEA was lower than that of free

Lac (Fig 3), the storage stability during recycled use for

3 months was higher than that by free Lac. Once again, these

results verified that the immobilized enzymes in each CLEA

were stable, but this was not observed in the case of free

enzymes. Although similar reusability of immobilized trypsin

on polymer nanofibers30 or CLEAs that were prepared using the

original procedure5,14 were previously reported, the reusability of

Fig. 3 pH profile of Lac-CLEA that was prepared by different con-

centrations of PA (%, v/v) and GA (%, v/v). The results are presented as

the activity recovery to free Lac at pH 4.5. All assays were conducted at

30 1C for 10 min. The concentration of free Lac was 5 mg mL�1. The

graph shows the mean � standard error for at least three experiments.

Fig. 4 Enzymatic activity of CS-CLEA. The results are presented as

the relative activity to that at 30 1C (black). The reactions were

measured at 50 1C (grey) and at 30 1C with 3 M urea (white). All

assays were performed in 20 mM Tris–HCl (pH 7.5). The concentra-

tion of free CS was 1 mg mL�1. The graph shows the mean � standard

error for at least three experiments.

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poly-Lys supported CLEA was better than or comparable to

those of the reported enzyme immobilization methods.

Conclusion

We studied the preparation of poly-Lys supported CLEAs

and their operational stability. Poly-Lys was added to form

co-aggregates with enzyme with a low Lys residue content. The

present immobilization procedure was achieved within 3 h

without the necessity for any special equipment. An acidic

enzyme can interact easily with basic poly-Lys, even at low

poly-Lys concentration, while a neutral or basic enzyme

requires high concentration of poly-Lys (Fig. S1 and S4, ESIw).Moreover, this method is applicable to the multi-subunit

protein (Fig. 4). In contrast, results of Lac-CLEA (Fig. 3)

illustrate the difficulty of the application for the enzyme

containing metal atom(s), indicating that the immobilization of

the metalloenzyme must be modified in terms of its procedure.

The immobilized enzymes in present CLEAs were more

stable against high temperature, pH, urea and DMSO than

free enzymes (Fig. S2, ESIw and Fig. 2 and 4). It is indicated

that the poly-Lys supported immobilization maintains their

configurations for their own enzymatic activity from denatura-

tion by heat or chemical reagents. In addition, CLEAs were

recycled without appreciable loss of activity, which is impor-

tant to ensure the cost-effective use of valuable enzymes. These

advantages suggest that the poly-Lys supported CLEA are

useful as catalysts in the fields of chemistry and biotechnology.

Acknowledgements

Part of this work was supported by Grant-in-Aid for Basic

Scientific Research (B: 20310074 and 23310092) from the

Japan Society for the Promotion of Science (JSPS).

Notes and references

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