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Enzyme and Microbial Technology 49 (2011) 446– 452

Contents lists available at ScienceDirect

Enzyme and Microbial Technology

j our na l ho me p age: www.elsev ier .com/ locate /emt

nhancement of a bacterial laccase thermostability through directedutagenesis of a surface loop

asrin Mollania, Khosro Khajeh ∗, Bijan Ranjbar, Saman Hosseinkhaniepartment of Biochemistry and Biophysics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran

r t i c l e i n f o

rticle history:eceived 13 February 2011eceived in revised form 28 July 2011ccepted 6 August 2011

eywords:

a b s t r a c t

Laccases (benzenediol oxygen oxidoreductases, EC 1.10.3.2) are used in many biotechnological processes,including removal of polyphenols in beverages, decolorizing and detoxifying effluents, drug analysis andbioremediation. In the present work, we have tried to increase thermal stability of laccase from Bacil-lus HR03 using site directed point mutations. Glu188 was substituted with 2 positive (Lys and Arg) andone hydrophobic (Ala) residues. All mutations showed improved thermal stability. Thermal activation of

accaseite-directed mutagenesishermostabilityhermal activation

laccase was also increased after introducing the mutations. Remarkably, the Glu188Lys variant showed3-fold higher thermal activation and higher T50 (5 ◦C) with respect to the native enzyme. Furthermoresteady-state kcat and Km values were influenced despite the distance between the mutated position andthe catalytic site. In Glu188Arg mutation, the kcat was improved 3-fold and Km reduced by 25%. Interest-ingly, all three variants showed higher stability against urea as a chemical denaturant. Structural analysesof the native and mutated variants were carried out using fluorescence and far-UV circular dichroism.

. Introduction

Enzymes intended for practical purposes need higher stabilitynd long-term activity under various unfavorable environmentalonditions (temperature, composition of solution and pH). There-ore a number of protein engineering efforts have been used torovide proteins with higher stability. Thermal stability as an

mportant characteristic of industrially used enzyme has been paidreat attention and factors enhancing protein thermostability haveeen extensively reviewed by the study of thermophilic proteins inomparison with their mesophilic counterparts [1–3].

Laccases (monophenol, dihydroxyphenylalanine: oxygen oxi-oreductase, EC 1.14.18.1) are copper-containing enzymes thatatalyze oxidation of polyphenols, polyamines and certain inor-anic ions beside reduction of molecular oxygen to water [4,5].hese enzymes show a typical three-domain fold with T1 mononu-lear copper centre in domain 3 and a trinuclear copper clustertwo T3 and one T2 copper ions) located between domains 1 and 3.

short loop followed by an �-helical fragment connects domains

and 2 whereas a large loop links domains 2 and 3 [6,7]. Till now

imited information is available regarding the effect of site-directedmino acid substitutions on the stability of bacterial laccases in

∗ Corresponding author at: Department of Biochemistry, Faculty of Biologicalciences, Tarbiat Modares University, P.O. Box 14115-175, Tehran, Iran.el.: +98 21 88009730; fax: +98 21 88009730.

E-mail address: khajeh@modares.ac.ir (K. Khajeh).

141-0229/$ – see front matter © 2011 Elsevier Inc. All rights reserved.oi:10.1016/j.enzmictec.2011.08.001

© 2011 Elsevier Inc. All rights reserved.

the presence of critical inactivating factors such as temperature,chemical denaturants and organic solvents. Several factors includ-ing surface distribution of charged residues, domain packing andprolin content have been proposed as important factors involvedin increased thermostability of some laccases [8].

Following previous studies on laccase isolated from local Bacil-lus sp. HR03, here we have tried to increase its thermal stability byapplying single mutations [9,10]. Sequence analysis of the isolatedgene showed highest similarity to the thermostable laccase (CotA)from Bacillus subtilis [11]. A three-dimensional model of laccasewas built using Swiss model utilizing CotA (1GSK) as the template(Fig. 1). The model showed superior presence of negatively chargedresidues on the surface regions of laccase from Bacillus HR03, whileCotA shows a positive patch at the interface between domains1 and 2. The significance of this positively charged patch is stillunknown [6]. On the other hand, previous studies have proposeddomain packing as a major factor related to laccase thermostabil-ity. Larger number of hydrophobic interactions between domains1 and 2 is responsible for higher degree of domain packing in theseenzymes. Fig. 1A represents the superimposed view of the inter-face loop between domains 1 and 2 in crystal structure of 1GSKand predicted structure of isolated laccase. All residues of the con-necting loop were analyzed using Eris and I-Mutant servers. Amongcandidates Glu188 showed two Tyr residues (189 and 250) and a

glutamate (246) as the neighbors. Since juxtaposition of phenolicrings between these neighboring residues might produce spatialhindrance or negative charge repulsion, Glu188 was selected asthe site of mutagenesis. The mentioned residue was substituted

N. Mollania et al. / Enzyme and Microbial Technology 49 (2011) 446– 452 447

Fig. 1. (A) Superimposition of the aminoacid residues located at the interface region of domains 1 and 2 from CotA (black) and laccase of Bacillus sp. HR03 (grey). (B) Ribbondiagram of the modeled laccase from Bacillus sp. HR03 based on crystal structure of CotA (PDB code: 1GSK), showing the location of the Glu188 in the surface loop betweend

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n two directions. First, the negatively charged Glu188 was substi-uted with Lys and Arg since the thermostable CotA shows higherresence of positive charges at the same region. Second, Glu 188as also mutated to Ala as a small hydrophobic amino acid in order

o increase the hydrophobic interactions which subsequently pro-ide higher level of domain packing in mutated enzyme. The effectsf these substitutions on the catalytic efficiency, thermal and chem-cal stability of the mutated enzymes were analyzed here. Also thetructural modifications were investigated using far-UV CD and flu-rescence techniques in comparison to the native enzyme as theontrol.

. Materials and methods

.1. Chemicals, bacterial strains and plasmids

2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), syringaldazineSGZ) and Isopropyl-�-d-thiogalactopyranoside (IPTG) were purchased from SigmaSt. Louis, MO, USA). Restriction endonucleases and dNTPs were obtained fromermentas (Germany). Molecular biology kits were from Bioneer (Korea). Pwo poly-erase prepared from Roche. All other chemicals were purchased from Merck

Darmstadt, Germany) and with analytical grade purity.

.2. Mutation design and site-directed mutagenesis

Among laccases which three-dimensional structures have been already solved,acillus sp. HR03 shares 98% sequence identity with CotA from B. subtilis (PDB code:GSK). The modeling was carried out by submitting the deduced protein sequencef laccase to the Swiss Model server (http://swissmodel.expasy.org//SWISS-ODEL.html). Three pairs of primers were designed in order to replace the Glu188ith 3 different residues. Site directed mutagenesis was performed with the Quick-hange-method described by Fisher and Pei [12]. PCR products were incubated withpnI restriction endonuclease at 37 ◦C for 16 h in order to degrade original DNA tem-lates. The reaction mixtures were transformed to Escherichia coli XL1-blue [13]. Theorrectness of substitutive mutations was confirmed after sequencing.

.3. Expression and purification of laccase

E. coli BL-21 cells harboring the native or mutated laccase genes were grownvernight at 37 ◦C in 25 ml Luria-Bertani (LB) medium containing ampicillin100 mg/ml). The overnight pre-culture was inoculated into fresh 1 l culture medium1% inoculation) containing the same antibiotic and incubated at 37 ◦C with shak-ng (180 rpm) until reached an OD600 of 0.5–0.6. Then, IPTG and CuSO4 were addedo a final concentration of 0.1 mM and 2 mM, respectively and temperature waseduced to 18 ◦C. The shaker was turned off 4 h after induction. The cells were har-ested by centrifugation after incubated for an additional 20 h at 18 ◦C without

haking. The pellet was resuspended in 100 mM potassium phosphate buffer, pH.6 containing PMSF and disrupted by sonication. Supernatant was heated at 70 ◦Cor 15 min and denatured proteins were removed by centrifugation. The remainingupernatant was loaded on Q-Sepharose column (Amersham Biosciences) that hadeen equilibrated before with 20 mM potassium phosphate buffer, pH 7.

2.4. Enzyme activity and biochemical characterization

Laccase activity was measured spectrophotometrically at room temperature.Oxidation of 0.05 mM SGZ (SGZ; 4-hydroxy-3,5-dimethoxybenzaldehyde) and2 mM ABTS (2,2′-azino-bis (3-ethylbenzathiazoline-6-sulfonate)) were followed in100 mM potassium phosphate and sodium citrate buffer pH 7.0 and 4.0, respectively.The increase in absorbance was measured at 525 nm (ε = 65,000 M−1 cm−1) for SGZ,and 420 nm (ε = 36,000 M−1 cm−1) for ABTS [14,15]. Steady-state kinetic parame-ters Km, Vmax, kcat and kcat/Km for mutants were determined and compared with thenative enzyme.

2.5. Thermal stability

Thermal stability was determined at three elevated temperatures (65, 70 and80 ◦C) in 100 mM potassium phosphate buffer (pH 7.0). After incubating each puri-fied enzyme for duration of 10–300 min in each temperature, tubes were chilled onice and the residual laccase activity was measured with SGZ and ABTS as substrate.A constant protein concentration was used for all thermal stability experiments inorder to avoid the effects of protein concentration on laccase stability. The stabil-ity of mutated variants was also quantified by determining T50, the temperature atwhich 50% of the initial laccase activity is retained after 30 min incubation comparedto the native enzyme.

2.6. Urea induced denaturation

Chemical denaturation of the enzyme was performed by incubating the enzymesin 100 mM potassium phosphate buffer, pH 7 in various concentrations of urea. Theactivity of the native protein in the absence of urea was determined as 100%.

2.7. Circular dichroism studies

Circular dichroism measurements were performed using a Jasco spectropo-larimeter J-715 (Tokyo, Japan). Far-UV CD (200–250 nm) was monitored with theenzyme concentration of 0.2 mg/ml in 100 mM potassium phosphate buffer, pH 7.Results were expressed as molar ellipticity [�] (deg cm2 dmol−1), based on a meanmolecular weight of the residue (MWR). Mean residue mass ellipticity was deter-mined 113 as the average molecular mass/residue. The molar ellipticity [�] wascalculated from the formula [�]� = (� × 100MRW)/(cl), where c is the protein con-centration in mg/ml, l the light path length in centimeters, and � the measuredellipticity in degrees at wavelength �. The parameters of the secondary structureswere calculated using S-715 CD-JASCO software.

2.8. Fluorescence measurements

The intrinsic fluorescence of the native enzyme and its variants was measuredon a PerkinElmer luminescence spectrometer LS 55. The excitation wavelength wasset at 280 nm and the emission spectra were recorded from 300 to 400 nm. All exper-iments were carried out at room temperature with protein concentrations of 20 �Min 100 mM potassium phosphate buffer, pH 7.0.

Fluorescence quenching experiments were carried out after addition of acry-lamide solution (1 M) to the protein (20 �g/ml) at pH 7.0. The final concentration ofacrylamide was variable between 0 and 200 mM with the incubation time of 5 min.The enzyme was excited at 280 nm and the emission spectra were scanned between300 and 400 nm. The decrease of fluorescence intensity at �max of emission was

448 N. Mollania et al. / Enzyme and Microbial Technology 49 (2011) 446– 452

Fig. 2. The kinetic parameters of the native and mutated variants were determined spectrophotometrically by measuring the oxidation of SGZ. (A) Native form, (B) Glu188Lysm x values were calculated from Michaelis–Menten curves using GraphPad Prism software.T

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Table 1Kinetic parameters of recombinant laccase variants with substitutions in Glu188.Activity was measured at room temperature for all samples using SGZ as a reducingsubatrate.

Mutant kcat (s−1) Km (�g/ml) kcat/Km

(s−1 �g−1 ml)

Recombinant laccase 20.0 ± 1.0 4.0 ± 0.2 5.0E188K 14.0 ± 0.7 5.5 ± 0.3 2.5

utated variant, (C) Glu188Arg and (D) Glu188Ala mutated variant. The Km and Vma

he standard errors were less than 6% of the experimental values.

nalyzed according to the Stern–Volmer equation: F0/F = 1 + Ksv [Q], where F0 and Fre the fluorescence intensities at the emission �max in the absence and presence ofuencher, [Q] is the quencher concentration, and Ksv is the quenching constant [16].

1-Anilino naphthalene-8-sulfonate (ANS) binding studies were performed in PerkinElmer luminescence spectrometer LS 50B. The spectra were measured inotassium phosphate buffer pH 7.0. The final concentrations of enzyme and ANS

n the enzyme solutions were 20 �g/ml and 30 �M, respectively. The ANS emissionas scanned between 400 and 600 nm after excitation at 380 nm.

. Results and discussion

.1. Production, purification and kinetic parameters

Low temperature and microaerobic conditions were used tomprove the production of laccase in E. coli BL21 cells [10]. Thenzymes were purified by ionic-exchange chromatography using-Sepharose columns. The purified wild type enzyme and its vari-nts showed a molecular mass of 65 kDa.

Kinetic analyses of three purified mutants (Glu188Lys,lu188Arg and Glu188Ala) are shown in Fig. 2. Wild-type laccasehowed kcat and Km values of 20 s−1 and 4 mM, respectively. Ashown in Table 1, despite of the distance between the mutatedesidue and active site of the enzyme, Glu188Arg mutation haslevated laccase catalytic efficiency through increased kcat andecreased Km values. A 25% reduction of Km value was observed forGZ as the substrate. Reduction in Km reflects the change in molec-lar recognition of substrate by laccase. As shown in Fig. 3A, in theative form of the laccase, Glu188 might be involved in salt bridges

ith Arg-248 and Lys-340 hence replacing this residue with any of

he amino acids will disrupt these connections. In the case of Argubstitution a new salt bridge between Arg188 and Glu246 can beormed (Fig. 3B). Disruption of old bridges along with formation of

E188R 60.0 ± 3.0 3.0 ± 0.2 20.0E188A 18.0 ± 0.9 3.5 ± 0.2 5.1

new bonds may affect the shape or size of the binding pocket. Inthe case of Glu188Arg, the binding pocket might have changed intoa potentially more favorable binding site, since kcat and kcat/Km ofthis variant improved by 3- and 4-fold, respectively. The increase ofKm and reduction of kcat were observed in Glu188Lys substitution.In the case of Glu188Ala, no significant changes were observed inthe enzymatic properties of the laccase. This single mutation seemsto impose minimal effect on the binding and oxidative ability of areducing substrate.

Generally, the kcat value for laccase oxidative reactions dependson the reduction rate of the T1 copper site. The donor–acceptorelectronic coupling, the reorganization energy and the redox poten-tials of the enzyme could affect this parameter [17–19]. Based onthese results, the increased catalytic efficiency which is observedfor Glu188Arg mutation might be due to reduction of reorga-nization energy or increase of redox potential and feasibility of

internal electron transfer rate. Lower kcat of the Glu188Lys muta-tion reflects an unfavorable situation for reduction and electrontransfer between substrate–enzyme Cu sites in this variant com-pared to the wild type.

N. Mollania et al. / Enzyme and Microbial Technology 49 (2011) 446– 452 449

Fig. 3. Local protein structure of laccase. The position of Glu188 and neighboring residues in the wild-type (A) and Glu188Arg variant (B) are shown. Residues are displayedin stick formats. The salt bridges are shown with a black line.

0

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ig. 4. Thermostability analysis of wild type and mutated laccase variants in potubstrate and (C) 70 ◦C for ABTS as the substrate (�: wild-type; �: Glu188Lys; �: Glalues.

.2. Thermostability of wild type and laccase mutated variants

The increased thermal resistance of the mutated laccasenzymes was confirmed after comparing the rate of thermal inac-ivation for wild type and its variants. As shown in Fig. 4A–D,ild-type and mutated variants exhibited different thermostability

n three temperatures (65, 70, 80 ◦C). Among these substitutions,lu188Lys showed the highest heat-resistance in all tested tem-eratures. Thermal stability of Glu188Arg and Glu188Ala showed alight improvement compared to the wild-type. The T50 was mea-ured for three enzyme variants and all of them possessed higher50 value. Since the laccase isolated from Bacillus sp. HR03 showshermal activation after 50 min heating at 70 ◦C, the mutated vari-

nts were also tested for this kind of activation. Results proved thathermal activation also happens in mutated variants after heat-ng. In Glu188Lys mutation, highest activation was observed inll three temperatures. Laccase activity was increased 9-fold after

phosphate buffer pH 7. (A) 65 C, (B) 70 C, (D) 80 C using SGZ as the reducingla and �: Glu188Arg). The standard deviations were within 5% of the experimental

temperature treatment at 70 ◦C with respect to the untreated con-trol enzyme. In addition, this substitution showed highest thermalstability.

In order to reveal possible reasons for higher stability ofmutated variants, adjacent amino acids near Glu188 were analyzed(Fig. 5A–D). Two phenolic groups of two Tyr residues and a negativeglutamate are juxtaposing this amino acid in the native conforma-tion. The spatial hindrance also repulsion energy possibly madebetween electronic clouds of phenolic groups and negative chargeof glutamic acid possibly has resulted in lower stability of the nativeform. Substitution of Glu 188 with Lys or Arg which has imposedpositive charge instead of the initial negative charge in this location,subsequently leads to formation of a new cation–� interaction. In

the case of Glu188Lys, the stability has increased dramatically whilelower stabilization was observed for Glu188Arg mutation. A torsionpossibly produced by the bulky side chain of Arg is suspected forlower stabilizing effect of this residue. In addition, based on WHAT

450 N. Mollania et al. / Enzyme and Microbial Technology 49 (2011) 446– 452

F 188 and neighboring residues of the wild-type laccase (A), Glu188Lys (B), Glu188Arg (C)a 0 are displayed in stick formats.

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100

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E188R

E188K

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ig. 5. Local structure of the wild-type laccase and its variants. The position of Glund Glu188Ala mutant (D) are shown. Residues 188, 189, 190, 191, 246, 250 and 34

F Web server, Arg substitution reduces the amount of hydrogenonds in comparison to Glu188Lys variant (data not shown).

.3. Mutation induced changes in laccase conformation

In order to analyze possible conformational changes inducedy mutations in laccase, we have used far-UV CD and fluores-ence techniques. In circular dichroism studies laccase variantsith substitutions in Glu188 showed no significant alterations in

he amount of secondary structures compared to the native formFig. 6A).

As shown in Fig. 6B, the intrinsic fluorescence of the Glu188Lysnd Glu188Ala substitutions was lower than the native enzymehile Glu188Arg variant showed higher emission. Fluorescence

mission of a protein is the result of its intrinsic fluorophores likerp and Tyr residues. The intensity of emission is related to therotein conformation which may expose or burry the internal fluo-ophores. The intrinsic quenching of a protein also might affect theuorescence intensity. Structural alterations which lead to dissoci-tion of quenchers will increase fluorescence intensity. In laccase,he type 3 copper with an absorption band at 330 nm may representhe intrinsic quenching effect in this enzyme.

In order to confirm whether the reduction of fluorescence inten-ity was the result of higher compactness of the structure oreduction of the quenching effect, more analyzes were carriedut using acrylamide quenching. Quenching of tryptophan fluores-ence by acrylamide was performed for the wild-type and variants.esults are shown in Fig. 7A. A linear Stern–Volmer plot is gen-rally indicative of a single class of aromatic residues, all equally

ccessible to acrylamide. The KSV values reported for the wild-typend variants are small, reflecting the buried position of aromaticesidues. Based on Stern–Volmer plots minor decrease of flexibil-ty was observed in Glu188Lys and Glu188Ala mutants compared to

Fig. 6. Structural modification analysis of the wild-type laccase and its variantsusing circular dichroism and intrinsic fluorescence. (A) Comparison of circulardichroism spectra between the native and mutated enzymes shows decreased sec-ondary structure. (B) Fluorescence intensities of laccase variants were increased incomparison to the wild type enzyme.

N. Mollania et al. / Enzyme and Microbial Technology 49 (2011) 446– 452 451

0

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Fig. 7. Acrylamide quenching and ANS binding analysis of the native laccase andits variants at room temperature. (A) Stern–Volmer plots of laccase fluorescencequenching (�: wild-type; �: Glu188Lys; �: Glu188Ala; �: Glu188Arg). The stan-dard deviations were within 5% of the experimental values. (B) Fluorescence spectraof wild-type and mutated laccase in the presence of 30 �M ANS. Results showedsignificant change in flexibility of the enzyme in the case of Glu188Arg mutation.

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40

80

120

160

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Fig. 8. The activity of laccase and its variants was measured at room temperature in

he native form. Therefore the lower intrinsic fluorescence in theseubstitutions might be the result of higher intrinsic quenchinggainst aromatic residue emission due to compactness of the struc-ure. As motioned above, the increase in thermostability and Km

alue of Glu188Lys variant may also confirm the improved struc-ural compactness, since some stabilizing mutations have impairedctive-site flexibility [20].

In the case of Glu188Arg variant, data revealed lower flexibil-ty in the structure of the enzyme. This substitution may decreasehe flexibility of the loop located between domains 1 and 2 ofhe enzyme. Also intrinsic fluorescence analysis confirmed thenhancement of emission. For more investigations, absorbanceas measured at 330 nm in order to find any changes happened

round T3 copper ions. In Glu188Arg substitution, 50% reductionf absorbance was observed compared to the native form withhe same concentration. Based on these results it is supposed thatigher emission of intrinsic fluorescence could be the result ofore tightness in the structure of the enzyme after elimination

f quenching effect of T3 copper ion. ANS binding studies werelso carried out. ANS is mainly non-fluorescent in aqueous solution

ut its emission intensity increases in hydrophobic environments21]. In the Glu188Arg variant, ANS binding analysis showed minoreduction in ANS emission resulted from slightly reduced bindingf ANS to hydrophobic patches (Fig. 7B).

100 mM phosphate buffer (pH 7) and urea concentration within the range of 1–6 M,using SGZ as a reducing substrate (�: wild-type; �: Glu188Lys; �: Glu188Ala and�: Glu188Arg). The standard deviations were within 5% of the experimental values.

3.4. Effect of urea on activity

Chemical denaturation of the native enzyme and its variants wasexamined in the presence of urea as a common denaturant. Fig. 8shows the effect of urea on the activity of laccase and its variants atroom temperature. The results presented in Fig. 8 show a definitedecrease in the rate of catalysis for all native and mutated vari-ants; the reducing rates of catalysis for mutated variants are not asfast as the observed amounts for the native enzyme in comparableconcentrations of urea. Interestingly in low concentrations, ureaactivated the mutated enzymes; the activity was enhanced pro-gressively with increasing concentrations of urea upto 0.1 M. Theactivation effect was slightly different for Glu188Arg, Lys and Alaby 1.08-, 1.3- and 1.5-fold, respectively. Higher aqueous concen-trations of this denaturant decreased the activity of laccase and itsvariants. Based on the activity assays variants retained 9, 10.5 and11% of their original activity in 6 M urea respectively, indicating thatthe mutated enzymes were partially unfolded but still functional.Therefore, all Glu188 substitutions have also increased denaturantstability of mutated enzymes compared to the native form. Chemi-cal denaturants such as urea might interact with proteins either in adirect manner or by modifying the behavior of solvents in their highconcentrations [22]. In mutated variants, it seems that low concen-trations of urea does not destroy but stabilizes protein structure.The stabilizing effect of urea has been previously reported. It hasbeen shown that urea can bind to protein and stabilize partiallyunfolded polypeptide chains hence enable the enzyme to maintaina more active conformation [23,24].

4. Conclusion

Modeling a thermostable Bacillus laccase, a negative residue atthe interface of domains 1 and 2 was substituted with two positiveresidues. Directed mutagenesis of the negative Glu188 to Arg simul-taneously improved laccase function also moderately increasedits compactness as fluorescence and acrylamide quenching exper-iments showed. Substitution of the same negative charge withlysine as another positive residue dramatically elevated thermalstability as well as thermal activation of this variant. The activity

of this mutated laccase increased by 9-folds following temperaturetreatment which is remarkably higher than the comparable amountfor the wild type enzyme. Structural compactness of this variantshowed remarkable promotion. The modified kinetic behavior of

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52 N. Mollania et al. / Enzyme and M

his variant might be the result of higher tightness of the enzymetructure after introducing this mutation. Introducing Ala at theame position with the aim of inducing higher domain packing andncreased hydrophobicity did not modify the kinetic parameters.owever, thermal stability of the variant increased compared to

he native enzyme.In summary, introducing positive charge in the connecting loop

etween domains 1 and 2, we were able to promote thermal sta-ility of laccase from Bacillus sp. HR03. Comparison of the twoositive substitutions (Glu188Lys and Glu188Arg) shows that notnly the reduction of negative charges can affect laccase stabilityut also the size of newly created positive residue might be impor-ant. Furthermore, our results from Glu188Ala prove that reductionf charged residues at the mentioned interface of domains mightncrease domain packing of the enzyme in accordance to previouseports.

cknowledgements

The authors express their gratitude to the Iran National Scienceoundation (grant no. 86013/03) and the research council of Tarbiatodares University for the financial support during the course of

his project.

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