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Biochemical Engineering Journal 49 (2010) 414–421

Contents lists available at ScienceDirect

Biochemical Engineering Journal

journa l homepage: www.e lsev ier .com/ locate /be j

mmobilization of PLP-dependent enzymes with cofactor retention andnhanced stability

armen López ∗, Sergio D. Ríos, Josep López-Santín, Gloria Caminal, Gregorio Álvaroepartament d’Enginyeria Química, Unitat de Biocatàlisi Aplicada associada al IQAC (UAB-CSIC), Escola d’Enginyeria Edifici Q,niversitat Autònoma de Barcelona, Campus de Bellaterra, E-08193 Bellaterra (Cerdanyola del Vallès), Catalunya, Spain

r t i c l e i n f o

rticle history:eceived 29 May 2009eceived in revised form 22 January 2010ccepted 9 February 2010

eywords:LP-dependent enzymesmmobilizationerine hydroxymethyltransferase

a b s t r a c t

Immobilization of PLP-dependent enzymes requires specific studies due to their special cofactor-enzymebond. These enzymes were immobilized using different methods, and the recombinant serine hydrox-ymethyltransferase (SHMT) was used as a case study. The immobilization of SHMT on glyoxal-agaroseresulted in a high retention yield (70%); however, the reduction step caused an enzymatic activity lossof 80%. The immobilization on Eupergit® C was optimized by considering different ionic strengths, pHand temperatures. SHMT reached 53% retention on this support. Although the enzymatic activity of thederivative decreased by 36% during the treatment with methylamine and washing, it was totally recov-ered by incubation with the cofactor. SHMT immobilized on Eupergit® C gained thermal stability withrespect to the soluble enzyme. Finally, 6-His-tagged SHMT was adsorbed very rapidly on IMAC supportsand reached a 98% immobilization yield and enzymatic retention. The capacity of Eupergit® C beads

lanine racemase

spartate aminotransferase to immobilize PLP-dependent enzymes was corroborated by the immobilization of alanine racemaseand aspartate aminotransferase. The final immobilization yields were 85 and 74% respectively, and the

d nin

derivatives were two- an

. Introduction

Pyridoxal phosphate (PLP)-dependent enzymes have been theubject of extensive research due to their catalytic versatility.he role of this coenzyme is very significant as it is involved inlarge number of reactions concerning amine and amino acidetabolism, including biosynthesis of amino acids and amino

cid-derived metabolites, and reactions involving biosyntheticathways of amino sugars and other amine-containing compounds.hese enzymes are able to catalyze racemization, decarboxylation,-elimination, replacement and transamination, among other reac-

ions [1,2]. Furthermore, some of these PLP-dependent enzymesave been identified as potential targets for therapeutical agents

n human diseases as well as herbicides [3]. The PLP group actss a cofactor in these enzymatic reactions. This cofactor is cova-ently bound to the enzyme by means of a Schiff base (internalldimine) between the aldehyde group of the PLP and the �-aminoroup of a lysine residue of the protein (Fig. 1). The amine-

ontaining substrate displaces the lysine amino group from thenternal aldimine, and in the process forms a new Schiff base withLP (external aldimine) [4]. PLP in the absence of enzyme enablesany of the possible reactions to occur slowly. The function of the

∗ Corresponding author. Tel.: +34 935812694; fax: +34 935812013.E-mail address: carmen.lopez@uab.es (C. López).

369-703X/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.bej.2010.02.004

e-fold more stable than the soluble enzymes.© 2010 Elsevier B.V. All rights reserved.

enzyme is to enforce substrate and reaction specificity as well asto enhance catalytic power [1]. The absence of PLP entails a lossof the enzymatic activity, although the incubation of the apoen-zyme with PLP allows the enzyme to recover the initial activity[5].

Immobilization of enzymes allows them to be easily recoveredand reused. It usually improves the stability of the enzyme and facil-itates process performance, thus enhancing cost saving and processproductivity. Immobilization to solid carriers is perhaps the mostused strategy, as it not only allows the retention of the enzyme butalso usually provides stability enhancements. In order to find themost appropriate technique, a set of supports has to be analyzed,as the behaviour of the enzyme on the support is very difficult topredict [6].

Immobilization of PLP-dependent enzymes has been reportedin very few works [3,7–9]. Tyrosine phenol-lyase and tryptophanindole-lyase were encapsulated in wet nanoporous silica gels [9]and �-transaminase was immobilized onto chitosan beads [7].In both cases moderate to low yields were obtained. The Schiffbase between the cofactor and enzyme is critical for enzymaticactivity and should be preserved during immobilization of PLP-

dependent enzymes. Thus, immobilization methods that involvereducing agents, such as NaBH4, should be avoided, as they irre-versibly deactivate the enzyme because the Schiff base establishedbetween PLP and the enzyme is reduced [10] (e.g. enzyme immo-bilization on glyoxal-agarose supports [11,12]).

C. López et al. / Biochemical Engineering Journal 49 (2010) 414–421 415

tion c

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2

2

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Fig. 1. General mechanism of the reac

The covalent immobilization of enzymes without further reduc-ion of the formed bonds can be performed by considering otherupports. Eupergit® C (epoxy-activated acrylic beads) has beenescribed as a good support for covalent attachment as it is verytable and has good chemical and mechanical properties [13].poxy-activated beads are bead polymers formed from a hydropho-ic acrylamide with allyl glycidyl (epoxide) groups as the activeomponents responsible for binding. The immobilization occurs viaucleophilic groups of the enzyme, which form extremely stableonds with the support. After immobilization, the excess of epoxyroups of the matrix must be blocked in order to avoid furthereactions [14,15].

Although covalent attachment of enzymes to a solid support hashe advantage that the enzyme cannot be desorbed from the sup-ort, the immobilization process is sometimes accompanied by aevere enzyme inactivation. In these cases, immobilization meth-ds based on affinity can be considered. In a previous work [16], arocedure for one-step purification and immobilization of recom-inant His-tagged DHAP-dependant aldolases by affinity on Co2+

helated supports (IMAC) was reported, and this approach coulde extended to PLP-dependent enzymes.

The main aim of this work is to evaluate and compare dif-erent methods for immobilizing PLP-dependent enzymes. Thenzyme serine hydroxymethyltransferase (SHMT; E.C. 2.1.2.1) haseen selected as a case study. In a previous work, the produc-ion, purification and biochemical characterization of Streptococcushermophilus SHMT cloned and expressed in E. coli were reported17]. This enzyme was obtained as a fusion protein with a his-idine tag and employed in different stereoselective syntheticeactions of aldol addition [17,18]. SHMT derivatives obtainedy covalent coupling will be compared with those prepared byffinity on metal-chelate supports in terms of PLP retention andtability. With the aim of providing a more general value to theesults and extending the immobilization methodology to otherLP-dependent enzymes, the procedures obtained were expandedo the immobilization of aspartate aminotransferase (AAT; E.C..6.1.1), an enzyme that we are investigating for the synthesis ofhiral amines, and alanine racemase (AR; E.C. 5.1.1.1).

. Materials and methods

.1. Chemicals and enzymes

Alanine racemase (AR) from Bacillus stearothermophilus, aspar-ate aminotransferase (AAT) from porcine heart, alcohol dehydro-enase (ADH) from Saccharomyces cerevisiae, l-alanine dehydroge-

atalyzed by PLP-dependent enzymes.

nase from Bacillus subtilis and malic dehydrogenase from bovineheart were obtained from Sigma Chemical Company (Munich, Ger-many). Agarose 6BCL gel beads were supplied by Iberagar (Coina,Portugal). Eupergit® C was obtained from Fluka (Munich, Ger-many). The IMAC supports were Chelating Sepharose® Fast Flowanion exchangers, from Amersham Pharmacia Biotech (Barcelona,Spain). All other chemicals were of analytical grade and were pur-chased from Sigma Chemical Company (Munich, Germany).

2.2. Serine hydroxymethyl transferase (SHMT) production

The glyA gene encoding a serine hydroxymethyl transferasewith treonine aldolase activity was isolated from Streptococcusthermophilus YKA-184 chromosomal DNA. The enzyme was over-expressed in Escherichia coli M15 as a recombinant protein witha His6-tag at its N-terminus and purified as previously described[17]. The specific activity of pure SHMT was 1.2 U/mg.

2.3. SHMT apoenzyme preparation

SHMT enzyme, precipitated in ammonium sulfate, was cen-trifuged at 12,000 rpm and 4 ◦C for 5 min in a Heraeus Biofuge Frescocentrifuge (DJB Labcare Ltd, Buckinghamshire, UK). After dissolvingthe solid in sodium bicarbonate 100 mM at pH 10, l-cysteine wasadded at different concentrations (0, 50, 100, 200, 400 mM) andincubated for 20 min. The activity loss in the presence of cysteinewas followed during this time. The separation of the l-cysteine–PLPcomplex from the apoenzyme was performed by size exclusionchromatography. The gel filtration experiments were carried outon a 20 cm3-column packed with Shephadex G-25 (GE Healthcare,Canada) on a FPLC system (Pharmacia, GMI; MN, USA). The columnwas equilibrated and run on 100 mM sodium bicarbonate buffer,pH 10. The SHMT-cysteine samples (200 �L) were loaded on thecolumn and run with a flow rate of 1 mL/min. Finally, the capacityof the apoenzyme for activity recovery was analyzed by incubationof 0.36 U/mL (0.3 mg/mL) with PLP cofactor at different concentra-tions up to 6 mM. Samples were taken from the solution containingPLP after 3 and 20 h and activity was analyzed as described below.

2.4. Covalent immobilization on glyoxal-agarose gels

Glyoxal-agarose 6BCL gels (agarose–O–CH2–CHO) were pre-pared by etherification of agarose gels with glycidol (2,3-epoxypropanol) and further oxidation with sodium periodate [19].The amount of aldehyde groups formed after 35 min of incubationwas measured by means of an enabled reaction using KI 10% and

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16 C. López et al. / Biochemical Eng

aturated NaHCO3. The number of aldehyde groups available to linkhe ligand was calculated by analyzing the consumed periodate andesulted in 240 �mol of aldehyde groups per mL of support.

In the immobilization process, a solution of NaHCO3 at pH 10as mixed with either SHMT or apoenzyme solution, leading to anal concentration of 100 mM NaHCO3 and 0.92 mg/mL of protein19]. An appropriate volume (9 mL, corresponding to 10 U or 8.3 mgf protein) of this solution was added to 1 mL of the activated sup-ort and 1 mL was used as a reference (blank) to test soluble enzymetability under immobilization conditions. The catalytic activity (inhe case of SHMT) or protein concentration (in the case of apoen-yme) of the blank, supernatant and total suspension was measuredt different times. The Schiff bases were reduced by adding a NaBH4oncentration of 1 mg/mL and shaking for 30 min [20]. After reduc-ion, both the immobilized apo and holoenzyme were separatedrom the supernatant by vacuum filtration, washed with water, fil-ered again and incubated in PLP solution (2-3-8 mM). Finally, thenzyme derivative was washed repeatedly with water, filtered andtored at 4 ◦C.

The immobilization yields were calculated from the enzy-atic activity measurements, as the difference between the initial

ctivity and the activity in the supernatant at the end of the immo-ilization process. In the case of the apoenzyme, immobilizationield was calculated from the protein measurements. The retainedctivity was obtained from the difference between the activity inhe suspension and the supernatant in relation to the initial activityf the process.

.5. Covalent immobilization on Eupergit® C

Eupergit® C beads were hydrated by incubation in 50 mM phos-hate buffer at pH 7.5 and 4 ◦C for 24 h. The immobilization of SHMTas carried out by mixing 1 mL of the wet support with 9 mL of

nzymatic solution containing 11.4 U (9.5 mg of protein). AnothermL containing the buffer and the enzyme at the same concentra-

ions were used as a blank. The effects of ionic strength (100 mMnd 1 M), pH (7.5 and 10) and temperature (4 and 25 ◦C) were stud-ed using phosphate and bicarbonate buffers as presented in Table 3discussed in the Section 3). The catalytic activity of the blank,upernatant and suspension was determined during the process.fter immobilization, the beads were collected by filtration underacuum using a porous glass filter. The excess of epoxide groupsn the matrix was blocked by incubation with 20% methylamineydrochloride or 0.5 M glycine in 50 mM potassium phosphate atH 8 and 4 ◦C for 24 h. The derivative was then washed with immo-ilization buffer and incubated in 2 mM PLP at 4 ◦C for 55 h. Finally,he beads were filtered, washed with buffer and dried before stor-ge at 4 ◦C.

Immobilization of AR on Eupergit® C was performed followinghe same protocol, offering 13 U (1.18 mg of protein) per mL of gelnd using 1 M KH2PO4 buffer at pH 7.5 and 1 M NaHCO3 buffer atH 10. AAT (9.9 U or 0.04 mg per mL of gel) was immobilized in theresence of 1 M KH2PO4 buffer at pH 7.5.

.6. Affinity immobilization on IMAC supports

The chelating sepharose, with iminodiacetic acid (IDA) as func-ionalized residues, was activated with cobalt. The support wasashed with three volumes of distilled water, incubated in three

olumes of 0.2 M CoCl2 at pH 4.7 for 12 h and washed again with fiveolumes of distilled water. Nine millilitres of SHMT solution con-

aining 9.2 mg of protein (either pure enzyme or supernatant fromellular lysate) in 20 mM phosphate buffer at pH 7.5 was mixedith 0.6 mL of support and another 1 mL was used as a reference

blank) [16]. The immobilization mixture was stirred in a horizon-al roller at 4 ◦C. Samples of the blank, suspension and supernatant

ng Journal 49 (2010) 414–421

were withdrawn and activity was tested. Finally, the solid deriva-tive was separated from the supernatant by filtration, washed withwater, incubated with 2 mM PLP and stored at 4 ◦C.

2.7. Thermal stability

The thermal stability assays were performed at 25 and 45 ◦C inan aqueous medium (200 mM potassium phosphate at pH 6.5 forSHMT; 200 mM sodium bicarbonate at pH 10 for AR; and 200 mMpotassium phosphate at pH 7.5 for AAT) starting from the same ini-tial activity for both the free and immobilized enzymes. Samples ofbiocatalyst were periodically removed and the activity measured.The pseudo half-life time was defined and calculated as the periodof time for the enzymatic activity to decrease by half.

2.8. Enzymatic activity measurements

SHMT immobilization was followed by measuring its threo-nine aldolase activity by means of a combined enzymatic reactionwith alcohol dehydrogenase [17,21]. l-Threonine is converted toglycine and acetaldehyde by SHMT, and acetaldehyde is subse-quently reduced to ethanol by ADH by means of the oxidationof NADH to NAD+. The assay mixture comprised 50 �mol of l-threonine, 100 �mol of potassium phosphate pH 6.5, 0.05 �mol ofPLP, 0.15 �mol of NADH, 30 U of ADH and appropriate amounts ofenzyme, in a final volume of 1 mL. The concentration of NADH wasmeasured at 37 ◦C at a wavelength of 340 nm in a UV–vis Cary spec-trophotometer (Varian, Palo Alto, CA, USA). The molar extinctioncoefficient of NADH at 340 nm is 6.22 mM−1 cm−1 [22,23]. One unitof threonine aldolase activity was defined as the amount of enzymethat catalyzed the formation of 1 �mol of acetaldehyde (1 �mol ofNADH oxidized) per minute at pH 6.5 and 37 ◦C.

AR activity was determined by a combined reaction with alaninedehydrogenase [24,25]. AR catalyzes the racemic conversion of d-alanine to l-alanine, which is subsequently converted to pyruvateand NH3 by l-alanine dehydrogenase by means of the reduction ofNAD+ to NADH. The concentration of this further compound wasfollowed by spectrometry. The reaction mixture contained a finalconcentration of 80 mM CAPS buffer (pH 10.5), 30 mM d-alanine,0.15 U/mL l-alanine dehydrogenase, 2.5 mM NAD+ and up to 50 �Lof sample in a total volume of 1 mL. Measurements were carried outat 37 ◦C at a wavelength of 340 nm. One unit of activity is defined asthe amount of enzyme required to catalyze the reaction of 1 �molof d-alanine to l-alanine per minute at pH 10.5 and 37 ◦C.

AAT activity was followed by a combined reaction withmalic dehydrogenase. L-aspartate transamination by AAT producesoxalacetate, which is reduced to malate by malic dehydrogenasewhile NADH is oxidized to NAD+. The reaction mixture contained afinal concentration of 75 mM phosphate buffer (pH 7.5), 193 mM L-aspartate, 12 mM �-ketoglutarate, 0.6 U/mL malic dehydrogenase,0.2 mM NADH and up to 50 �L of sample in a total volume of 1 mL.Measurements were carried out at 37 ◦C at a wavelength of 340 nm.One unit of activity is defined as the amount of enzyme requiredto catalyze the reaction of 1 �mol of L-aspartate to oxalacetate perminute at pH 7.5 and 37 ◦C.

When suspension samples were tested, magnetic stirring wasapplied to the spectrophotometric cuvette. The relative error for theenzymatic activity method was around ±4% for soluble enzymesand less than ±10% for suspension samples.

2.9. Protein concentration

The concentration of proteins was determined by the Bradfordmethod, using 1 mL of Coomassie® protein assay reagent pur-chased from Pierce (Rockford, IL, USA) and 20 �L of sample. After10 min the absorbance at 595 nm was read in a UV–vis Cary spec-

ineering Journal 49 (2010) 414–421 417

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Table 1Activity recovery after incubation of the SHMT apoenzyme with PLP. The values ofenzymatic activity (EA) are referred to the initial activity (EA0), before removing thecofactor.

PLP (mM) Activity recovery (EA/EA0)

0 h 3 h 20 h

0 0 0 00.125 0.60 0.65 0.650.25 0.75 0.60 0.650.5 0.60 0.65 0.701 0.80 0.65 0.704 0.75 0.65 0.706 0.70 0.65 0.80

Enzyme activity relative error: ±4%.

Table 2Activity recovery after PLP incubation of SHMT apoenzyme immobilized on glyoxal-agarose. The values of enzymatic activity (EA) are referred to the initial activity (EA0),before removing the cofactor.

PLP (mM) Activity recovery (EA/EA0)

0 h 24 h 120 h

2 0.10 0.09 0.09

Fs

C. López et al. / Biochemical Eng

rophotometer (Varian, Palo Alto, CA, USA) and the concentrationas calculated from a calibration with standard samples of bovine

erum albumin.

. Results and discussion

Streptococcus thermophilus serine hydromethyltransferase wasxpressed in E. coli and produced and purified as previouslyublished [17]. The recombinant enzyme was provided with a 6-istidine tag, which facilitates the purification and recovery steps.

.1. SHMT immobilization by covalent attachment onlyoxal-agarose

The immobilization of enzymes by covalent attachment onlyoxal-agarose requires the further reduction of the Schiff baseonds formed. In the case of PLP-dependent enzymes, this pro-ess also results in the reduction of the internal aldimine betweenLP and Lys, thus preventing the Schiff base exchange reactionnd product release, and consequently causing enzymatic activityosses [26–28]. In order to avoid the reduction of the PLP-enzymeond, the cofactor was removed from the enzyme before the

mmobilization process. It has been previously reported that SHMTncubation with L-Cys allows a stable thiazolidine complex to beormed between the amino acid and the cofactor [29,30]. Therefore,he presence of L-Cys displaces the Schiff base PLP enzyme; this isccompanied by the disappearance of the characteristic absorbancet 430 nm and the appearance of an increase at 330 nm in the UVpectrum [28]. Purified SHMT was incubated with different L-Cysoncentrations and the removal of PLP was followed by monitoringhe decrease in absorbance (data not shown). Most of the enzymectivity (95%) was found to be lost after 20 min of incubation in00 mM L-Cys solution. The apoenzyme was purified from the L-ys incubation mixture by gel filtration chromatography. Beforepoenzyme immobilization, its ability to recover activity after incu-ation with PLP cofactor was assayed at different concentrations.he results, presented in Table 1, show immediate activity recoveryn all cases, and values between 60 and 80% of the initial enzy-

atic activity (before PLP removal) are achieved. The variations areithin the limits of standard error of the measurement. The enzy-

atic activity test was not influenced by the presence of additional

LP.SHMT apoenzyme was immobilized by covalent attachment on

glyoxal-agarose support at pH 10, which ensures unprotonatednd therefore very reactive amino groups in the protein [31]. Pro-

ig. 2. Immobilization course of SHMT apoenzyme (A) and active SHMT (B) on glyoxaluspension. Curves through data points are to help visualize the time courses. Enzyme ac

3 0.12 0.11 0.088 0.12 0.09 0.14

Enzyme activity relative error: ±4% soluble, and ±10% immobilized derivative.

cess evolution was followed by measuring the total protein in thesupernatant compared with a blank control (Fig. 2A). All the offeredprotein was immobilized in less than 1 h. The activity of the immo-bilized apoenzyme could not be determined due to the absenceof cofactor. For comparison, a parallel experiment was performedwith the active enzyme (containing PLP). In this later experiment,SHMT activity was measured in both the supernatant and suspen-sion, and the results are presented in Fig. 2B. As in the former case,practically all the offered protein was immobilized, and the activ-ity recovered in the derivative was around 70%. Nevertheless, asexpected [30], 80% of the offered units were lost after the reductionof the derivative with sodium borohydride. A further incubationwith PLP, even as much as 10 mM, only enabled the enzyme torecover 25% of the initial enzymatic activity.

The immobilized apoenzyme was also submitted to sodium

borohydride reduction and assayed for activity recovery by PLPincubation. Even employing a PLP:enzyme concentration ratio of500:1, only a slight activity percentage was recovered (Table 2). Itcould be hypothesized that, as the attachment between the glyoxal-

-agarose in 100 mM NaHCO3 at pH 10 and 25 ◦C. (©) Blank; (�) supernatant; (N)tivity relative error: ±4% soluble, ±10% immobilized derivative.

418 C. López et al. / Biochemical Engineeri

Tab

le3

Effe

ctof

the

imm

obil

izat

ion

con

dit

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son

the

imm

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dan

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ew

ith

outs

up

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he

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izat

ion

con

dit

ion

s.

Enzy

me

Sup

por

tB

uff

erC

once

ntr

atio

n(M

)p

HT

(◦ C)

Imm

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tim

e(h

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mob

iliz

atio

nyi

eld

(%)

Ret

ain

edac

tivi

ty(%

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ctiv

ity

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nal

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a(%

)A

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ity

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Pin

cuba

tion

(%)

Fin

alac

tivi

tyof

blan

k(%

)

SHM

TG

lyox

al-a

garo

seN

aHC

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0.1

1025

110

070

2025

100

SHM

Tap

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zym

eG

lyox

al-a

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seN

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0.1

1025

110

00b

0b14

100

SHM

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per

git®

CN

aHC

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0.1

1025

9635

15n

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.10

0SH

MT

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31

1025

2490

18n

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0SH

MT

Eup

ergi

t®C

KH

2PO

41

7.5

2570

9353

3450

100

SHM

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per

git®

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H2PO

41

7.5

416

075

50n

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n.d

.10

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027.

54

198

9888

100

100

Lysa

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ith

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-Co

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027.

54

110

098

8898

100

AR

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41

7.5

2524

9685

3568

91A

REu

per

git®

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aHC

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110

254

9891

5185

84A

AT

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41

7.5

2510

9866

4974

84

n.d

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tive

erro

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e,±1

0%im

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ativ

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The

fin

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the

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ases

wit

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he

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ocki

ng

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mai

ned

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sw

ith

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nal

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five

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mat

icac

tivi

ty.

ng Journal 49 (2010) 414–421

agarose and enzyme occurs via amino groups of the enzyme, thesupport probably reacts with the lysine residue of the active site,thus blocking the cofactor access. On the other hand, the enzymerigidity conferred by the multipoint covalent attachment is some-times not beneficial for preserving the catalytic capacity of theenzyme. The reduction in protein flexibility can be a hindrance forconformational changes that take place during the catalytic cycle ofsome enzymes [8]. These low enzyme retention yields, added to thePLP consumption, limit the practical application of this derivativeas a biocatalyst.

3.2. SHMT immobilization by covalent attachment on Eupergit® C

SHMT immobilization through the epoxy groups of Eupergit® Csupports was investigated as an alternative procedure for covalentattachment without previous cofactor removal. The hydrophobic-ity of this support favours a two-step immobilization process:first, the hydrophobic surface area of the enzyme rapidly interactswith the hydrophobic carrier; and second, the covalent attachmentbetween the amino groups of the enzyme and the epoxy groups ofthe support takes place [32]. This procedure allows protein immo-bilization under mild conditions. After enzyme attachment, theremaining free epoxy groups have to be blocked by incubation withsuitable compounds. These compounds usually contain an aminogroup, and therefore they need basic pH to enhance their reactivity;some of the most commonly applied compounds are methylamineand glycine [14,15]. Pure SHMT in soluble form was incubated insolutions containing these components. Methylamine was selectedas the blocking compound of epoxide groups as the enzyme incu-bated in its presence at a concentration of 20% retained almost100% of the initial activity even after 72 h. Glycine, apart from beinga natural substrate for SHMT, was described to be an inhibitor ofthis enzyme [33]. Consequently, the enzyme lost 40% of its initialactivity after incubation with 0.5 M glycine for 72 h.

With the aim of optimizing the immobilization yield andretained activity percentage, several immobilization conditions[15,34] were assayed (Table 3). High buffer concentrations favouredand accelerated the immobilization process, since raising the ionicstrength of the solution increases the strength of hydrophobicinteractions [15]. Although the oxirane groups of Eupergit® C canreact with proteins over a wide pH range [15], the covalent bind-ing on these supports was faster at alkaline pH as the enzymehas more amino groups that can react with the support; there-fore, multipoint covalent immobilization is favoured. However,high activity loss was observed in the derivative when the pro-cess was performed at pH 10, while the activity of the blank wasmaintained at 100% at the same conditions. As previously indi-cated, covalent multipoint immobilization sometimes reduces thecatalytic capacity of the enzymes. Apart from the effects statedabove, the multiple attachments can contribute to the alterationof the conformational equilibrium between the open and closedforms of the enzyme [9,35]. For PLP-dependent enzymes, apolarenolimine and polar ketoenamine are two tautomers of the internalaldimine formed between PLP and the enzyme. When the equilib-rium enolimine–ketoenamine is displaced to the enolimine form,activity losses were reported for other PLP-depending enzymes(tryptophan synthase) [8]. Considering this possibility, a more neu-tral value was assayed for SHMT immobilization, which resulted ina slower but more activity retaining process. At pH lower than 7the reactivity of amino groups, and hence the immobilization yield,

decreases considerably. Finally, both immobilization rate and yieldbenefited from room temperature vs. refrigeration at 4 ◦C (Table 3).Under the best conditions around 90% of the offered enzymaticactivity was immobilized, retaining more than 53% of the initialoffered activity (11.4 U/mL of support).

ineering Journal 49 (2010) 414–421 419

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Fig. 3. Thermal stability of SHMT in 200 mM KH2PO4 at pH 6.5 at 25 ◦C (A) and45 ◦C (B). (O) Soluble SHMT; (©) SHMT immobilized on Eupergit® C; (�) SHMT

C. López et al. / Biochemical Eng

The unreacted epoxy groups of the support were blocked byncubation of the enzyme derivative with 20% (v/v) methylamine.t was observed that the enzyme lost around 36% of its activityfter methylamine incubation and washing with distilled waterTable 3). As no desorption of enzyme was observed, the activityecrease was attributed to a PLP loss during washing. In order toestore activity, the derivative was incubated in 2 mM PLP solu-ion. At neutral pH, the Schiff base formation between enzymend PLP was expected to be slower than at alkaline pH; there-ore, the incubation was maintained for 3 days. After 48 h, thectivity of the derivative before blocking was almost totally recov-red, and no more losses occurred even after several washingrocesses. The activity loss could be explained by the high affin-

ty between methylamine and PLP to form a Schiff base bond,hus displacing the bond between the cofactor and the lysinef the active site. The washing process performed after incuba-ion would sweep the PLP–methylamine complex, thus leavinghe inactive apoenzyme bound to the support. This mechanismas proposed in the literature to explain the inhibition of PLP-ependent enzymes in the presence of some inhibitory compounds36] and the formation of the apoenzyme by incubation with cys-eine [30].

.3. SHMT immobilization by adsorption to IMAC supports

The interest of the enzymatic immobilization on IMAC supportsies in the possibility of performing purification and immobiliza-ion of the enzyme from the supernatant of disrupted microbialulture in only one step. In this case, the enzyme must be ableo interact with the chelated metal of the support. Therefore,HMT was expressed as a 6-His-tagged protein as describedefore [17]. The use of the IMAC technique for soluble enzymeurification requires a further step of enzyme desorption and sup-ort recovery by means of incubation with imidazol, which hasore affinity for the chelated metal of the support and displaces

he protein. When the purpose is the simultaneous purifica-ion and immobilization of the enzyme, this second step is notequired.

Purified SHMT (11 U/mL) was immobilized on Co-IDA supportsnd the blank, supernatant and suspension activities were fol-owed. After less than 1 h all the offered enzyme was attached to theupport, reaching 98% of immobilization yield and 98% of retainedctivity (Table 3). The derivative was washed several times aftermmobilization, and lost 12% of the enzymatic activity. Incubation

ith 2 mM PLP for 24 h allowed the enzyme to recover the totalnitial activity; the activity decrease could be due to the PLP beingemoved during the washing process.

The feasibility of simultaneous purification and immobiliza-ion was assayed with SHMT produced from recombinant E. coliultures. The supernatant from cell lysate centrifugation was sub-itted directly to immobilization on Co-IDA supports. The time

ourse of the immobilization was very similar to that of pure SHMT:he process was completed after 1 h, and an immobilization yieldnd retained activity of almost 100% were obtained (Table 3). Afterve washes with water and incubation with 2 mM PLP, the enzymeaintained the initial activity. The treatment by IMAC supports was

emonstrated to be very useful for the simultaneous purificationnd immobilization of 6-His-tagged SHMT.

.4. Thermal stability of the enzymatic derivatives

One of the purposes of enzyme immobilization is to enhance thetability of the enzyme. One easy and low time-consuming way toest the improvement in stability is to compare the free and immo-ilized enzymatic activity decays at a high temperature. Thermaltability experiments were performed with free and immobilized

immobilized on IMAC support. The inset in (B) details the initial activity evolutionwith time. Curves through data points are to help visualize the time courses. Enzymeactivity relative error: ±4% soluble, ±10% immobilized derivative.

SHMT (Eupergit® C and IMAC derivatives), which were incubatedin buffer in the absence of substrate. The enzymes were placed attemperatures of 25 and 45 ◦C and the enzymatic activities weremeasured at different incubation times (Fig. 3). In both cases, asudden drop of enzymatic activity was observed for immobilizedenzymes in the first 10 h. Sometimes the protein surface has two ormore areas with reactive groups which facilitate bonding to thesupport. In many cases, immobilization by covalent attachmentgives a main orientation, but it is difficult to guarantee that it is theonly orientation of the enzyme. Attachment at different areas mayconfer different rigidity to the enzyme and subsequently differentdegrees of stability [6]. Diverse populations of SHMT were probablybound to the support by means of different groups of residues; theless stable ones lost the activity in the first hours, but the main pop-ulation maintained the activity for a longer period of time withoutan appreciable additional loss.

It can be highlighted that at both temperatures the free enzymelost activity much more rapidly than the immobilized enzymes.While at 25 ◦C the pseudo half-life time was around 100 h for thesoluble enzyme, after 400 h the derivatives retained more than 80%

of the initial activity (Fig. 3A). At 45 ◦C the soluble enzyme was veryunstable, whereas the enzyme immobilized on IMAC and Eupergit®

C retained 40 and 60% of the initial activity after 230 h respectively(Fig. 3B).

4 ineering Journal 49 (2010) 414–421

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20 C. López et al. / Biochemical Eng

The covalent attachment of the enzyme SHMT to the supporteems to confer more stability than the immobilization based ondsorption mechanisms. Any enzymatic immobilization inside aorous solid can enhance the operational stability of the enzymeecause the matrix may hinder the rotational freedom of the pro-ein, and hence the unfolding induced by any distorting agent (pH,eat, organic solvents) may occur to a lesser extent [6,37]. Theseossible conformational changes of the protein are more limitedhen the immobilization takes place by means of multipoint cova-

ent attachment, which contributes to the rigidity of the enzyme6].

.5. Immobilization and thermal stability of other PLP-dependentnzymes

Based on the SHMT immobilization results, Eupergit® C waselected as a suitable support for the immobilization of other PLP-ependent biocatalysts. AR and AAT were selected as proteins of

nterest, as they are commercially available and belong to two dif-erent classes of enzymes.

AR was immobilized at pH 7.5 and 10, as this enzyme waseported to be stable at high pH values, which favour the immo-ilization by amino groups [38]. For the immobilization at pH 7.5he retention of the enzymatic activity on the support reached 85%fter 24 h, whereas the immobilization at pH 10 permitted 91% ofhe initial activity to be retained after 4 h (Table 3). In this case,he attachment between AR and the support by a higher numberf points, favoured at alkaline pH, seems to result in an appro-riate enzyme orientation for preserving the catalytic capacitiesf the protein. AAT is poorly stable at basic pH; therefore, it wasmmobilized at pH 7.5, and reached 66.5% activity retention after0 h (Table 3). In both cases the non-reacted epoxy groups werelocked with methylamine and the derivatives were washed. Asor SHMT, the enzymes lost a considerable percentage of theirnzymatic activity during this treatment, although almost all thectivity was recovered after incubation with PLP (Table 3). Evenhough each enzyme can have different behaviour during immo-ilization, we can state that these results represent a substantial

mprovement with respect to the work by Yi et al. [7], who wereble to immobilize �-transaminase on Eupergit® C with only 11%ctivity retention. Moreover, Pioselli et al. [8,9] encapsulated PLP-ependent enzymes and obtained a six- to eight-fold decrease

n their catalytic activity. After rejecting the possibility of limita-ions of substrate diffusion, they hypothesized that the activity lossuring immobilization could be due to a decrease in the rate of con-ormational changes that accompany the catalytic cycle. In ordero avoid this activity loss, the authors considered a change of gel

atrix chemical properties. In all cases, a larger improvement innzymatic retention would be achieved by appropriately orient-ng the enzyme and the carrier, i.e., by controlling the protein areattached to the support; however, this “controlled immobilization”s still little developed [6].

The thermal stability of both AR and AAT derivatives wasssayed and compared to the free enzymes at both 25 and 45 ◦C.t the lower temperature, free and immobilized enzymes were

ully stable for several months. At 45 ◦C, the stabilizing effect of themmobilization was revealed (Fig. 4). The pseudo half-life time oflanine racemase increased from 34 h for the free enzyme to 60 h forhe enzyme immobilized on Eupergit® C (Fig. 4A). This increase wasigher for AAT, for which the pseudo half-life time increased almostine-fold by means of the immobilization (Fig. 4B). The different

evels of stabilization achieved by these two enzymes by means ofmmobilization could be due to the different natures of the residueshat are more exposed to covalent bonds with the support. AAT isossibly linked by a higher number of attachments, and this wouldonfer a better stability to the enzyme.

Fig. 4. Thermal stability at 45 ◦C of AR in 200 mM NaHCO3 at pH 10 (A) and AATin 200 mM KH2PO4 at pH 7.5 (B). (O) Soluble enzyme; (�) enzyme immobilizedon Eupergit® C. Curves through data points are to help visualize the time courses.Enzyme activity relative error: ±4% soluble, ±10% immobilized derivative.

4. Conclusions

A methodology for the preparation of stable and active immo-bilized PLP-dependent enzymes has been developed. Due to theparticular properties of the cofactor-enzyme bond, the covalentimmobilization on glyoxal-agarose supports could not be com-pleted successfully. However, Eupergit® C supports were shown tobe suitable for a multipoint covalent attachment of PLP-dependentenzymes. By optimizing operational conditions, immobilizationyields over 90% and retention yields over 50% were obtained for thethree PLP-dependent enzymes considered. The activity loss causedby blocking the unreacted groups of the support was recovered byincubation with PLP. Moreover, the covalent immobilization con-tributed extensively to the thermal stabilization of the enzymes andderivatives even nine-fold more stable than the free enzymes wereobtained. Although not all the populations of immobilized enzymewithstood high temperatures, a large percentage of catalyticactivity was maintained for a prolonged period of time. The immo-bilization by adsorption on IMAC supports resulted in higher immo-bilization yields and stable derivatives, although this option is onlysuitable for recombinant enzymes containing appropriate tags.

Acknowledgements

The Department of Chemical Engineering of the UAB consti-tutes the Biochemical Engineering Unit of the Reference Network

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C. López et al. / Biochemical Eng

n Biotechnology of the Generalitat de Catalunya (XRB). This workas supported by the Spanish Ministry of Science and Innovation

MICINN; project CTQ2008-00578/PPQ), the European Researchrea (ERA-IB; Eng Biocat project EUI2008-03615) and Generalitate Catalunya (DURSI 2005SGR 00698). Carmen López would like tohank the Spanish Ministry of Education and Science for a Juan dea Cierva postdoctoral contract.

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