Structure-Based Engineering of Methionine Residues in the CatalyticCores of Alkaline Amylase from Alkalimonas amylolytica for ImprovedOxidative Stability

Haiquan Yang,a,b Long Liu,a,b Mingxing Wang,a,b Jianghua Li,a,b Nam Sun Wang,c Guocheng Du,a,b and Jian Chend

Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, Chinaa; School of Biotechnology, Jiangnan University, Wuxi, Chinab;Department of Chemical and Biomolecular Engineering, University of Maryland, College Park, Maryland, USAc; and National Engineering Laboratory for CerealFermentation Technology, Jiangnan University, Wuxi, Chinad

This work aims to improve the oxidative stability of alkaline amylase from Alkalimonas amylolytica through structure-basedsite-directed mutagenesis. Based on an analysis of the tertiary structure, five methionines (Met 145, Met 214, Met 229, Met 247,and Met 317) were selected as the mutation sites and individually replaced with leucine. In the presence of 500 mM H2O2 at 35°Cfor 5 h, the wild-type enzyme and the M145L, M214L, M229L, M247L, and M317L mutants retained 10%, 28%, 46%, 28%, 72%,and 43% of the original activity, respectively. Concomitantly, the alkaline stability, thermal stability, and catalytic efficiency ofthe M247L mutant were also improved. The pH stability of the mutants (M145L, M214L, M229L, and M317L) remained un-changed compared to that of the wild-type enzyme, while the stable pH range of the M247L mutant was extended from pH 7.0 to11.0 for the wild type to pH 6.0 to 12.0 for the mutant. The wild-type enzyme lost its activity after incubation at 50°C for 2 h, andthe M145L, M214L, M229L, and M317L mutants retained less than 14% of the activity, whereas the M247L mutant retained 34%of the activity under the same conditions. Compared to the wild-type enzyme, the kcat values of the M145L, M214L, M229L, andM317L mutants decreased, while that of the M247L mutant increased slightly from 5.0 � 104 to 5.6 � 104 min�1. The mechanismresponsible for the increased oxidative stability, alkaline stability, thermal stability, and catalytic efficiency of the M247L mutantwas further analyzed with a structure model. The combinational mutants were also constructed, and their biochemical proper-ties were characterized. The resistance of the wild-type enzyme and the mutants to surfactants and detergents was also investi-gated. Our results indicate that the M247L mutant has great potential in the detergent and textile industries.

�-Amylases hydrolyze starch by cleaving �-1,4-glucosidic link-ages and have been used widely in the food, textile, and phar-

maceutical industries (10, 16, 20–22, 25). Alkaline �-amylaseshave high catalytic efficiency and stability at alkaline pHs from 9 to11 (5, 9, 11, 17) and have applications in the starch and textileindustries, where starch is hydrolyzed under alkaline conditions(5, 10, 16, 21, 22).

Oxidative stability is one of the most important quality param-eters for alkaline amylase, especially in detergents where the wash-ing environment is oxidizing (13). Methionine (Met) residues inproteins are especially oxidation prone (4, 27). The oxidation ofthis residue has been shown to cause a decrease in activity or anoutright inactivation of amylases (12, 23). To reduce inactivationcaused by oxidation, the replacement of methionine by an oxida-tive-resistant amino acid may be effective. The nonoxidizableamino acid residues include leucine (Leu), serine (Ser), isoleucine(Ile), threonine (Thr), and alanine (Ala). For example, Met 197 ofthe �-amylase from Geobacillus stearothermophilus US110 was re-placed with Ala, and the mutant retained 70% of the activity in thepresence of 1.8 M H2O2 after 60 min of treatment (15).

In previous work, the alkaline amylase (AmyA) from the alka-liphilic Alkalimonas amylolytica strain N10 was expressed in Esch-erichia coli DH5� (28). However, AmyA is susceptible to oxida-tion by hydrogen peroxide.

On the other hand, an oxidation-resistant AmyA could be animportant candidate for the combined desizing, bioscouring, andbleaching (CDBB) process in the detergent and textile industries.In this work, we aim to improve the oxidative resistance of thisenzyme via the site-directed mutagenesis of methionine residues.

Based on an analysis of a three-dimensional (3D) structure modelof the enzyme, five methionine residues (Met 145, Met 214, Met229, Met 247, and Met 317) around the active site of AmyA wereselected for mutation. These methionines were individually re-placed by leucine, and the mutant enzymes are designated theM145L, M214L, M229L, M247L, and M317L mutants, respec-tively. Combinational mutants with multiple mutation sites werealso constructed. The antioxidant and other biochemical proper-ties (alkaline stability, thermal stability, catalytic efficiency, andspecific activity) of the mutants were characterized and comparedwith those of the wild-type enzyme, and the possible mechanismsresponsible for changes in the biochemical properties were ex-plored by analyzing the structure model. Our mutagenesis proce-dure described here may be applied to improve the antioxidantabilities of other industrial enzymes.

MATERIALS AND METHODSStrains and plasmids. All strains and plasmids used were obtained fromthe Laboratory of Biosystem and Bioprocess Engineering at Jiangnan Uni-versity. The alkaline amylase gene (GenBank accession no. AY268953)

Received 22 April 2012 Accepted 10 July 2012

Published ahead of print 3 August 2012

Address correspondence to Long Liu, longliu@jiangnan.edu.cn, or Jian Chen,jchen@jiangnan.edu.cn.

Copyright © 2012, American Society for Microbiology. All Rights Reserved.

doi:10.1128/AEM.01307-12

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from alkaliphilic A. amylolytica N10 was synthesized by Sangon BiotechCo., Ltd. (Shanghai), based on the preferred codon usage of Escherichiacoli. Plasmid pET-20b(�) was used for subcloning, Escherichia coli JM109was the host for cloning work, and E. coli BL21(DE3) was the host for theexpression of the alkaline amylase.

Media and culture conditions. Luria-Bertani (LB) medium was usedfor the seed culture of E. coli. The E. coli bacteria were cultured on a rotaryshaker at 200 rpm at 37°C for 10 h. Terrific broth (TB) medium was usedfor the production of alkaline amylase. E. coli transformants carrying therecombinant plasmid were screened on corn starch-agar plates. After in-cubation at 37°C for 10 h, the formation of a clear halo around individualcolonies indicated the expression of the alkaline amylase induced by theinducer isopropyl-�-D-1-thiogalactopyranoside (IPTG) (0.4 mM) in theplate. Fermentation was performed with 250-ml shaker flasks containing25 ml medium, and the mixture was agitated at 200 rpm at 37°C; when theoptical density at 600 nm (OD600) reached 0.6, IPTG was added to 0.4 mMfor induction at 25°C for 48 h.

Site-directed mutagenesis. The recombinant plasmid was PCR am-plified with mutagenic oligonucleotides (Table 1) by using the Mutan-BEST kit (TaKaRa, Dalian, China). Fragments amplified by PCR werepurified and isolated on 1% agarose gels after electrophoresis. The frag-ments were blunted by Blunting Kination enzyme mix (TaKaRa, Dalian,China) through the Blunting Kination reaction. The blunt-end fragmentswere ligated with ligation solution I (TaKaRa, Dalian, China). The reac-tion mixture was then transformed into competent cells of E. coli JM109.The transformants were selected at 37°C on LB agar plates containing 50�g/ml ampicillin. The alkaline �-amylase gene in the transformants wasconfirmed by PCR and checked by sequencing, and finally, the recombi-nant plasmids were transformed into competent cells of E. coli BL21(DE3)for expression.

Purification of alkaline amylase. Solid ammonium sulfate was addedto the E. coli supernatant to 70% saturation at 0°C. The precipitate wascollected and dissolved in glycine-NaOH buffer (pH 9.0, 20 mM) anddialyzed overnight against the same buffer. After dialysis, the enzymesolution was filtered. The enzyme solution was then injected into an Aktapurifier (GE Healthcare) through anion exchange (Q-Sepharose HP).Buffer A (equilibration buffer) contained 20 mM phosphate buffer (pH6.0). Buffer B (elution buffer) contained 20 mM phosphate buffer (pH6.0) and 1 M NaCl. The flow rate was 1.0 ml/min. When the sample wasinjected, buffer A was used to absorb the target enzyme to the column, andafter the unbound proteins were eluted, a linear elution was done byramping buffer B from 0% to 100%. The fractions were collected foractivity assays and sodium dodecyl sulfate (SDS)-PAGE analysis.

Enzyme assays. Alkaline amylase activity was determined by measur-ing the amount of reducing sugar released during the hydrolysis of 1%soluble starch in glycine-NaOH buffer (pH 10.0, 20 mM) at 55°C for 5min. A control without the addition of enzyme was used. The amount ofreducing sugar was measured with a modified dinitrosalicylic acidmethod (7). One unit of alkaline amylase activity was defined as theamount of enzyme that released 1 �mol of reducing sugar as glucose permin under the assay conditions. The protein concentration was measuredwith the Bradford method (28), with bovine albumin (Sangon Biotech,Shanghai, China) as the standard.

Computer-aided modeling of the tertiary structure. The theoreticalstructure of alkaline �-amylase was obtained by homology modeling withthe Swiss Model server (1, 8). Amino acid mutations were inserted into thestructure with the mutation tool of Swiss-Viewer, followed by side-chainreconstruction for neighboring amino acids and energy minimization (2).The root mean square deviation (RMSD) for the modeling structure wascalculated with Swiss-PdbViewer 4.0.4. The number of hydrogen bonds inthe enzyme was calculated with Discovery Studio 2.5.

Influence of mutations on oxidative stability. The wild-type and en-gineered enzymes were incubated with 500 mM H2O2 in glycine-NaOHbuffer (pH 9.0, 20 mM) at 35°C for 5 h. The choice of the H2O2 concen-tration was dictated by that (approximately 500 mM) typically employedin the bleaching process in the textile industry when combined with de-sizing and bioscouring (17). After the 5-h H2O2 incubation period, cata-lase (10,000 U/ml) was immediately added to the samples to a final con-centration of 2,000 U/ml to quench the remaining H2O2. The startingconcentration for alkaline �-amylase was 16 U/ml when incubated withH2O2. Samples (200 �l) were then withdrawn to measure the residualactivity under standard assay conditions.

Effect of temperature and pH on stability. The thermal stability ofalkaline �-amylase was determined at 30°C to 50°C in glycine-NaOHbuffer (pH 9.0, 20 mM). The enzyme was incubated at 30°C for 10 h andwas then incubated at 40°C or 50°C for 2 h. The activation energy (Ea) wasdetermined in the temperature range of 20°C to 50°C by the Arrheniusequation: ln(k) � (�Ea/RT) � ln(A) (where R is the mole gas constantand T is the thermodynamic temperature). The reaction rate constant (k)was calculated from the equation ln(c) � �kt � ln(c0), where c is theconcentration of the substrate (soluble starch) and c0 is the initial concen-tration of the substrate. To estimate the optimal pH of alkaline �-amylase,the purified protein was incubated in sodium phosphate buffer (pH 6.0 to8.0, 20 mM), Tris-HCl buffer (pH 8.0 to 9.5, 20 mM), glycine-NaOHbuffer (pH 9.5 to 11.0, 20 mM), and Ca(OH)2 buffer (pH 12.0, 20 mM).The pH stability of alkaline �-amylase was determined at pHs rangingfrom 6.0 to 12.0 at 25°C for 24 h. After incubation, the alkaline �-amylaseactivity was measured at pH 10.0 and at 55°C. The starting concentrationfor alkaline �-amylase was 16 U/ml.

Influence of mutations on kinetic parameters. The kinetic parame-ters (Km, Vmax, kcat, and kcat/Km) of the wild-type enzyme and mutantswere determined in glycine-NaOH buffer (pH 10.0, 20 mM) at 55°C.Assays were performed with active enzyme at a fixed starting level of 16U/ml and soluble starch at concentrations ranging from 1 to 10 g/liter.The enzyme kinetic parameters Km and Vmax were estimated from Eadie-Hofstee plots (6).

Effects of surfactants and detergents on stability and activity. Theeffects of the surfactants Tween 20, Tween 60, Tween 80, Triton X-100,and SDS on enzyme stability were studied by preincubating the enzyme at40°C for 1 h. The concentration of surfactants was 10% (wt/vol). Thestarting concentration for alkaline �-amylase was 16 U/ml. Residual en-zyme activity was measured under standard assay conditions. The activityof the enzyme without the addition of surfactant was taken as 100%. Thecompatibility of the enzyme with solid detergents was studied using solidlaundry soap (Nice, China), toilet soap (Safeguard), washing powder 1(Tide), and washing powder 2 (Nice, China). The compatibility of theenzyme with liquid detergents was studied using laundry detergent 1(Blue Noon, China), laundry detergent 2 (Liby, China), liquid detergent 1(Nice, China), and liquid detergent 2 (Liby, China). The detergents werefirst diluted in tap water to give concentrations of 70 mg/ml for soliddetergents and 10% for liquid detergents. The endogenous proteases fromthe detergents were inactivated by the incubation of the detergents at 65°Cfor 1 h before addition. When enzyme activity was determined, the deter-gents were diluted to give final concentrations of 7 mg/ml for solid deter-gents and 1% for liquid detergents to simulate washing conditions (15).The enzyme activity of the control without the addition of detergent wastaken as 100%.

TABLE 1 Oligonucleotide primers used for site-directed mutagenesis

Enzyme Nucleotide sequence (5=¡3=)a

M145L CCATCGTATG(CTG)GGTGCGGM214L TATCTGATG(CTG)GGTGAAGACGTTGACTM229L AGGAGATG(CTG)AAGGCGTGGGM247L TTTCGTATG(CTG)GACGCGATTGCM317L CGGAGATATG(CTG)CGTTGGTGCGGa Nucleotides underlined correspond to the codons chosen for mutation. Nucleotides inparentheses replaced the underlined nucleotides.

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CD. Circular dichroism (CD) spectra were measured on a MOS-450/AF-CD-STP-A instrument (Bio-Logic, France) at a protein concentrationof 0.1 mg/ml in a 1-cm-path-length quartz cuvette in 20 mM Tris-HClbuffer (pH 9.0). In order to minimize signal baseline drift, the spectropo-larimeter and xenon lamp were warmed up for at least 30 min prior toeach experiment. Ellipticity data for the enzymes in the band of 190 to 250nm were collected, from which the spectrum of a buffer blank was sub-tracted.

RESULTS AND DISCUSSIONSelection of mutation sites based on structure analysis. The ox-idative stability of enzymes is one of the most important parame-ters for their use in the presence of bleach-containing detergents intextile processing. Previous studies of the oxidative inactivation ofsome industrial enzymes indicated that the oxidation of methio-nine residues to their sulfoxide derivatives is a main problem (27).The oxidation of methionine residues situated in the cavity of theactive site increases the size of the side chain, leading to the sterichindrance of the active site (15).

To determine the mutation sites of alkaline amylase, a 3D (three-dimensional) model of alkaline amylase was constructed based on theknown structure of �-amylase B (3bc9) by using the Swiss Modelserver (Fig. 1A) (26). The amino acid sequence of alkaline amylaseincludes four conserved regions (regions I through IV) that were pre-viously identified in the �-amylase family: 137DVVFNH142 (regionI), 244GFRMDAIAH252 (region II), 278EAWV281 (region III), and335FVDNHD340 (region IV) (28). The four conserved regions arenecessary for catalytic activity, and they are present in the catalyticdomain of the (�/�)8-barrel structure of �-amylases in family 13.

Region I contains three fairly conserved amino acids (Asp 137,Asn 141, and His 142) that are important for the stability andactivity of the enzyme. Region II contains the catalytic nucleophileresidue Asp 248 and invariant residue Arg 246. The two residuesare found in all �-amylases and are believed to be indispensablefor catalytic activity. Region III includes the catalytic proton do-nor Glu 278, which is the only totally conserved residue in thisregion. Region IV contains the only fully conserved residue, Asp340 (28). The Na/Ca binding site in AmyA corresponds to Asn142, Pro 211, and Asp 217, whereas the active site of AmyA in-cludes Asp 248, Glu 278, and Asp 340.

As suggested by the constructed model (Fig. 1), Met 145, Met214, Met 229, and Met 247 are situated in the cavity of the activesite, with the Met side chains pointing toward the substrate. Theoxidation of these methionine residues could increase the size ofthe side chain and cause a steric obstruction of the active site (15).Moreover, the replacement of these methionine residues with leu-cine may improve the antioxidative resistance of the enzyme toH2O2. Surface-exposed methionine residues in proteins were alsoshown to be susceptible to oxidation (14). Although Met 317 is notin the cavity of the active site, it is sufficiently close to the active siteand exposed to the surface (Fig. 1) and may also contribute to theoxidation stability of the enzyme. Thus, structure analysis ofthe enzyme suggests that these five methionine residues may bethe key amino acids for improving antioxidative properties.

Expression of mutant proteins. Based on the above-describedstructure analysis, five methionine residues were replaced withleucine through site-directed mutagenesis. After the verification

FIG 1 Model structure of alkaline amylase (A) and local model of mutation sites and catalytic residues in alkaline amylase (B). (A) The model structure of AmyAwas constructed with the crystal structure of AmyB (3bc9) as a template (26). The root mean square deviation (RSMD) of alpha carbon atoms for the modelingstructure was 33.76 Å. The �-helices and �-sheets are shown in red and cyan, respectively. The catalytic residues Asp 248, Glu 278, and Asp 340 are shown in a“CPK” (Corey-Pauling-Koltun) representation. The “sticks” indicate the mutant positions. The oxygen atoms are in red, the nitrogen atoms are in light blue, thecarbon atoms are in yellow-green, and the sulfur atoms are in yellow. (B) The distances from the respective catalytic residues to Met 145, Met 214, Met 229, Met247, and Met 317 in AmyA are shown. The oxygen atoms are in red for Met and dark purple for catalytic residues, the nitrogen atoms are in purple for Met anddark purple for catalytic residues, the carbon atoms are in green, and the sulfur atoms are in yellow.

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of the gene sequence, the mutant genes were recombined withpET-20b(�) and expressed in E. coli BL21(DE3). SDS-PAGEanalysis showed that the protein after mutation was approxi-mately 60 kDa (Fig. 2).

Oxidative stability of the wild-type and mutant proteins. Thewild-type and mutant enzymes were evaluated for their toleranceto H2O2. The wild-type enzyme was susceptible to oxidation, withless than 10% of the original activity being retained when theenzyme was incubated with 500 mM H2O2 at 35°C for 5 h (Fig. 3).In contrast, the M145L, M214L, M229L, M247L, and M317L mu-tants exhibited an enhanced oxidative tolerance to H2O2 and re-tained 28%, 46%, 28%, 72%, and 43% of the original activity,respectively, in the presence of 500 mM H2O2 for 5 h. It was notedthat the remaining activity of the M247L mutant was enhanced by7-fold compared with that of the wild-type enzyme.

The methionine residue of amylase plays an important role inoxidative sensitivity. The oxidation of methionine was shown pre-viously to cause an activity decrease or a inactivation of amylase(15). Thus, the replacement of the methionine residue with anantioxidative amino acid may improve the oxidative stability ofthe enzyme. For example, Met 197, which is situated close to theactive site of amylase from Geobacillus stearothermophilus, hasbeen shown to be involved in oxidative inactivation, and the in-troduction of a non-sulfur-containing amino acid at this positiongreatly reduced the oxidative sensitivity of the enzyme (15). Thiswork confirms the importance of Met 145, Met 214, Met 229, Met247, and Met 317 in oxidative sensitivity. As suggested by theconstructed model, Met 145, Met 214, Met 229, and Met 247 aresituated in the cavity of the active site, with the Met side chainspointing toward the substrate. Among these five methionines,Met 247 is closest to the catalytic residues (Fig. 1B). After Met 247was replaced by leucine, the enzyme became most resistant tooxidation in the presence of H2O2. Surface-exposed methionineresidues in proteins were also shown previously to be susceptibleto oxidation (14). Here, Met 317, not in the cavity of the active sitebut close to the active site and exposed to the surface, also showedincreased oxidation stability when replaced by leucine.

Effects of pH on the stability of mutant enzymes. The effectsof pH on the stability of the mutant enzymes were studied. Asshown in Fig. 4A, the stable pH range of the M145L, M214L,M229L, and M317L mutants remained unchanged compared tothat of the wild type. In contrast, the stable pH range of the M247Lmutant was extended from pH 7.0 to 11.0 to pH 6.0 to 12.0. Theresults were further confirmed by CD spectra (Fig. 5). The CDspectra of the wild-type and engineered enzymes from 190 to 250

nm at 25°C show that the secondary structure of the mutant en-zymes was not significantly altered compared with that of thewild-type enzyme.

In order to further explain the effect of mutations on pH sta-bility, the enzyme’s hydrogen bonding network was studied. Asshown in Fig. 6, the mutation at Met 247 affected the number andlocations of hydrogen bonds around the conserved regions of theenzyme. Mutation increased the number of total hydrogen bondsfrom 437 to 449, and it also increased the number of hydrogenbonds around the active site. The hydrogen bonding networkplays an important role in determining the pH stability of amylase(19), and a strengthened hydrogen bonding interaction may havecontributed to the increased pH stability after mutation in thiswork.

Effects of temperature on stability of mutant enzymes. Figure4B shows the effects of temperature on the stability of the wild-type and mutant enzymes. Both the wild-type and mutant en-zymes retained more than approximately 80% of their originalactivity after incubation at 30°C and at pH 9.0 for 10 h. The stabletemperature range of the mutant enzymes (except for the M247Lmutant) did not change compared to that of the wild-type en-zyme. The wild-type enzyme completely lost its activity after in-cubation at 50°C for 2 h, while the M247L mutant retained 34% ofthe original activity after incubation under the same conditions.In other words, the M247L mutant is more temperature tolerantthan the wild type. The activation energies (Ea) of the wild typeand the M145L, M214L, M229L, M247L, and M317L mutantswere calculated from the Arrhenius equation [ln(k) � (�Ea/RT) � ln(A)] and were determined to be 36.1, 78.4, 43.0, 52.4,37.3, and 38.1 kJ/mol, respectively. After mutation, the activationenergies of the mutants increased. The melting temperature of thisenzyme was 64.3°C, as determined by differential scanning calo-rimetry (DSC).

The secondary structure of the mutants changed little aftermutation by CD spectrum analysis (Fig. 5). However, the hydro-gen bonds appear to be responsible for maintaining enzyme sta-bility at high temperatures (4). At high temperatures, the enzyme

FIG 2 SDS-PAGE analysis of the purified wild-type and mutant proteins.Lanes: M, molecular mass marker; 1, the M145L mutant; 2, the M214L mutant;3, the M229L mutant; 4, the M247L mutant; 5, the M317L mutant.

FIG 3 Oxidative stability of wild-type (WT) and mutant proteins. The relativeactivity (percent) was determined and compared with the activity without theaddition of any H2O2. The mutant alkaline amylases and the wild type wereincubated with 500 mM H2O2 at 35°C for 5 h.

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seems to be unable to retain the tightly coiled, thermostable, andcatalytically active structure in the absence of proper hydrogenbonds (4). The replacement of Met by Leu caused changes in thehydrogen bonds, and the number of hydrogen bonds increasedfrom 437 to 449 (Fig. 6). The M247L mutant became more stableat high temperatures, possibly due to strengthened electrostaticinteractions within the enzyme.

Influence of mutations on kinetic parameters and specificactivity. Table 2 shows the kinetic parameters (Km, Vmax, kcat, andkcat/Km) and specific activities of the wild-type and mutant en-zymes. Compared with that of the wild-type enzyme, the Km val-ues of all the mutants decreased, and the Vmax values of theM145L, M214L, M229L, and M317L mutants decreased signifi-cantly, while that of the M247L mutant increased from 38.8 to 42.3�mol/(ml · min). The catalytic constants (kcat) of the M145L,M214L, M229L, and M317L mutants decreased by 57, 40, 32, and

1%, respectively, while that of the M247L mutant increased from5.0 � 104 to 5.6 � 104 min�1. Usually, this type of point mutationhas a significant influence on the catalytic efficiency of the enzyme.For example, the replacement of Met 197 in the Bacillus licheni-formis amylase with a nonoxidizable amino acid resulted in a sig-nificant decrease in the catalytic efficiency (23). Incidentally, herethe kcat/Km values of the M145L, M214L, M229L, M247L, andM317L mutants increased by 13, 73, 36, 113, and 34%, respec-tively. A mutation of methionine to leucine, which resulted in anincreased catalytic efficiency, was also reported in a previous work(18), where a 3.2-fold increase in the catalytic efficiency was ob-served upon the replacement of the corresponding methionineresidue (Met 208) by leucine in His6-tagged BLA�NC Bacillus sp.strain TS-23 (18). The wild-type and mutant enzymes displayedsimilar secondary structures based on far-UV CD spectrum anal-ysis (Fig. 5). The increased substrate binding ability (as indicatedby the decreased Km) of the mutants contributed to the increase ofthe kcat/Km ratio, while the increase in the substrate affinity mayhave resulted from changes in electrostatic interactions aroundthe catalytic residues after mutation (Fig. 4).

In most cases, site-directed mutagenesis has a negative influ-ence on specific activity. For example, the specific activity de-creased from 1,000 to 845 U/mg when Met 197 was replaced by Alain �-amylase from Geobacillus stearothermophilus US100 (15),and the specific activity greatly decreased when mutations wereintroduced at positions 315 and 446 in �-amylase from Bacillus sp.TS-23 (18). As shown in Table 2, the specific activities of theM145L, M214L, M229L, and M317L mutants decreased in thiswork, while that of the M247L mutant increased compared to thatof the wild-type enzyme.

Effects of surfactants and detergents on the stability and ac-tivity of mutant enzymes. To evaluate the mutants’ potential util-ity in the detergent and textile industries, the activity of alkalineamylase was evaluated in the presence of surfactants, includingTween 20, Tween 60, Tween 80, Triton X-100, and SDS. As shownin Fig. 7A, compared to that of the wild-type enzyme, the activityof the mutant enzymes decreased after incubation at 35°C for 1 h

FIG 4 Effect of pH and temperature on stability of alkaline amylase after mutation. (A) Effect of pH on stability of alkaline amylase after mutation. The relativeactivity (percent) was determined and compared with the activity that was highest at different pHs for the same enzyme. (B) Effect of temperature on stability ofalkaline amylase after mutation. The relative activity (percent) was calculated on the basis of the activity that was determined before heat treatment for eachenzyme (100%). The inset presents the Arrhenius plot of the logarithm of the k values against the reciprocal of the absolute temperature (T). The values shownare activation energies calculated from the plot.

FIG 5 The conformation change of the mutants was checked by circular di-chroism (CD) spectrum analysis.

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in the presence of 10% (wt/vol) Tween 60 or Triton X-100. Tween20 or Tween 80 slightly increased the activity of the M247L mutantcompared with the activity of the wild-type enzyme. Incubationwith SDS increased the activity of the mutant enzymes comparedwith the activity of the wild-type enzyme. Anionic surfactants

such as SDS may have altered electrostatic interactions with theenzyme, causing a change in the enzyme activity (3). For nonionicsurfactants such as Tween 20, Tween 60, Tween 80, and TritonX-100, without electrostatic interactions with the enzyme, the ef-fect on the enzyme activity might be due to local structural rear-rangements of the critical residues in the active site as a conse-quence of the mutations (3, 24).

An evaluation of the effect of surfactants alone on the mutatedenzymes is not sufficient in gauging suitability for their incorpo-ration into commercial detergents. Therefore, the activity of theenzymes in the presence of different solid and liquid detergentswas studied (Fig. 7B). The wild-type and engineered �-amylaseswere found to be less stable in the presence of liquid detergents. Inthe presence of laundry detergents or liquid detergents, the activ-ity of the enzymes decreased to less than 50%. However, the en-zymes were stable in the presence of solid detergents (washingpowder); the activity increased for some engineered enzymes. Inthe presence of washing powder, the activity of the M229L,M247L, and M317L mutants increased compared to that of thewild type. The addition of an engineered enzyme (M247L) intowashing powder 1 and washing powder 2 increased activities by35% and 30%, respectively, compared to the activities before theaddition of the powders. However, in the presence of solid soap,all of the wild-type and engineered enzymes were less stable. Theseresults suggest that after mutation, the M247L mutant becamemore compatible with the majority of solid detergents (washingpowders). This observation agrees well with other studies whereincubation in the presence of Lav� and Nadhif (detergents) in-creased the activity of AmyUS100�IG/M197A from Geobacillusstearothermophilus by 10 to 20% (15).

Effect of combinational mutations on oxidative resistanceand catalytic efficiency. In order to further verify the importance ofthe five methionines for oxidative tolerance, combinational muta-tions of the five sites were done, and the biochemical properties of theresulting mutants were characterized (Table 3). For double muta-tions, the oxidative tolerance improved for the M145/M247L (M145/247L), M214/247L, M229/247L, and M247/317L mutants, with theM145/247L mutant retaining 76.7% of the original activity in thepresence of 500 mM H2O2 after 5 h. For triple mutations, the oxida-tive tolerance also improved significantly for the mutants involvingM247, specifically with the M214/247/317L mutant retaining 87.7%of the original activity in the presence of 500 mM H2O2 after 5 h. It isinteresting that the oxidative stability of the M214/229/317L triplemutant was also greatly enhanced. For combinational four-site mu-tations, the M214/229/247/317L and M145/229/247/317L mutantscompletely lost activity after mutation, whereas the M145/214/247/317L mutant retained 91.2% of its original activity. For the combina-

FIG 6 Local hydrogen bonding network of the enzyme. The active site isshown in a purple CPK (Corey-Pauling-Koltun) representation. The “ball andstick” indicate the conserved regions. The sticks indicate other residues. Thegreen CPK representations indicate Met residues that are replaced by Leu inthis work. The dashed lines are shown for comparison of the different hydro-gen bonds of the wild-type enzyme to those of the M247L mutant after muta-tion. (A) The local hydrogen bonding network of the wild-type enzyme. (B)The local hydrogen bonding network after M247L mutation.

TABLE 2 Specific activities and kinetic parameters of wild-type and mutant alkaline amylases

EnzymeMean Km

(g/liter) SDa

Mean Vmax [�mol/(ml · min)] SD

Mean kcat

(103 min�1) SDb

Mean kcat/Km [103 liters/(g · min)] SD

Mean sp act (103

U/�mol) SD

Wild type 9.0 0.1 38.8 1.3 50.3 1.5 5.6 0.4 106.2 1.3M145L 3.4 0.1 19.2 1.4 21.4 1.3 6.3 0.8 55.5 1.9M214L 3.1 0.1 22.7 1.2 30.2 1.2 9.7 0.7 62.1 0.7M229L 4.5 0.2 27.2 1.1 34.1 1.9 7.6 1.1 74.7 3.2M247L 4.7 0.2 42.3 1.0 56.1 1.6 11.9 0.7 117.0 1.3M317L 6.6 0.2 36.3 2.6 49.6 2.9 7.5 0.8 98.1 5.2a km, substrate dissociation constant.b kcat in �mol dextrose equivalents per minute per �mol protein.

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tional five-site mutations, the activity of the M145/214/229/247/317Lmutant was completely lost. In general, the mutants involving Met247 exhibited a higher oxidative tolerance to H2O2. Thus, Met 247 isthe most critical residue for the oxidative stability of this enzyme.

The catalytic parameters of enzymes were determined for com-binational mutations in this work (Table 3). After mutation, theKm values of the M145/214L, M145/214/229L, M145/229/247L,M214/229/247L, and M145/214/229/317L mutants decreased

FIG 7 Effects of surfactants and detergents on the wild type and mutants. (A) Effects of surfactants on stability of the wild type and mutants. The relative activity(percent) was calculated on the basis of the activity for each enzyme which was determined without the addition of any surfactants (100%). (B) Effects ofdetergents on activity of the wild type and mutants. The relative activity (percent) was calculated on the basis of the activity that was determined without theaddition of any detergents (100%).

TABLE 3 Oxidative stability and kinetic parameters of wild-type and mutant enzymes after combinational mutationsd

EnzymeMean Km

(g/liter) SDa

Mean Vmax [�mol/(ml · min)] SD

Mean kcat (103

min�1) SDb

Mean kcat/Km [103 liters/(g · min)] SD

Mean sp act (103

U/�mol) SDMean oxidativestability (%) SDc

Wild type 9.0 0.1 38.8 2.1 50.3 1.5 5.6 0.4 106.2 1.3 10.0 1.4M145/214L 5.2 0.1 25.3 1.8 31.7 1.6 6.1 0.4 31.2 0.9 24.8 3.5M145/229L 12.5 0.2 17.2 1.9 8.7 1.2 0.7 0.1 15.2 1.1 23.8 2.3M145/247L 7.3 0.2 28.0 2.1 36.8 4.3 5.2 0.6 74.5 1.5 76.7 2.9M145/317L 7.2 0.1 15.0 1.2 24.2 2.4 3.4 0.4 57.2 3.2 39.2 2.0M214/229L 13.4 0.1 6.9 0.9 7.2 2.1 0.5 0.2 10.2 1.0 31.8 1.1M214/247L 26.4 0.3 147.0 3.2 208.9 4.6 8.1 0.4 92.1 2.4 72.0 1.2M214/317L 40.1 0.5 126.6 2.5 169.1 5.2 4.2 0.2 118.5 4.5 41.0 2.7M229/247L 10.2 0.1 47.4 2.2 58.3 2.2 5.7 0.3 96.1 2.6 65.0 3.3M229/317L 11.5 0.1 19.2 1.7 12.7 1.5 1.1 0.1 25.1 1.3 49.0 4.7M247/317L 10.1 0.3 26.1 2.0 33.5 1.9 3.4 0.4 38.7 1.8 74.0 4.1M145/214/229L 4.6 0.1 13.3 1.0 5.5 1.0 1.2 0.2 3.5 0.8 29.4 2.2M145/214/247L 13.2 0.5 47.6 2.3 39.7 3.4 3.1 0.5 34.1 2.0 74.1 1.3M145/214/317L 9.8 0.2 19.3 1.5 8.0 1.3 0.8 0.2 4.1 0.9 39.5 4.1M145/229/247L 3.7 0.1 5.4 1.1 2.3 0.8 0.6 0.3 2.1 0.4 75.1 3.6M145/229/317L 16.7 0.3 9.9 1.4 4.1 1.4 0.3 0.1 1.2 0.5 51.3 3.2M145/247/317L 8.8 0.2 3.5 0.8 1.5 0.5 0.2 0.1 0.9 0.3 40.1 1.4M214/229/247L 5.7 0.1 14.2 1.4 5.9 1.3 1.0 0.3 3.3 0.7 68.9 2.7M214/229/317L 6.2 0.1 1.7 0.6 0.7 0.2 0.1 0.0 0.6 0.1 96.5 5.2M214/247/317L 11.8 0.3 27.3 2.0 56.3 6.1 4.7 0.7 33.6 2.8 87.7 2.1M229/247/317L 5.0 0.1 18.2 1.7 37.9 3.1 7.5 0.9 78.9 2.4 72.1 2.7M145/214/229/247L 89.0 1.3 49.8 3.0 20.8 3.7 0.2 0.1 3.4 0.6 70.6 3.1M145/214/229/317L 2.7 0.4 7.3 1.1 3.0 1.0 1.1 0.6 2.0 0.5 73.2 2.5M214/229/247/317L — — — — — —M145/214/247/317L 14.4 0.9 39.2 2.5 81.7 2.2 5.7 0.5 33.0 2.8 91.2 4.3M145/229/247/317L — — — — — —M145/214/229/247/317L — — — — — —a Km, substrate dissociation constant.b kcat in �mol dextrose equivalents per minute per �mol protein.c The activity retained after the enzymes were incubated with 500 mM H2O2 at 35°C for 5 h.d —, the activity of the enzyme was lost.

Protein Engineering of Alkaline Amylase

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from 9.0 g/liter for the wild type to 5.2, 4.6, 3.7, 5.7, 5.0, and 2.7g/liter, respectively, for the mutants. Thus, these combinationalmutations are important for enhanced substrate binding. TheVmax values of the M214/247L, M214/317, M145/214/247L, andM145/214/229/247L mutants greatly increased from 38.8 �mol/(ml · min) for the wild type to 147.0, 126.6, 47.6, and 49.8 �mol/(ml · min), respectively, for the mutants. The interaction of thesemutation sites is critical for the reaction rate of the enzyme. Thecatalytic constants (kcat) of the M214/247L, M214/317L, andM145/214/247/317L mutants increased from 5.0 � 104 min�1 forthe wild type to 20.8 � 104, 16.9 � 104, and 8.2 � 104 min�1,respectively, for the mutants. The catalytic efficiency, indicated bythe kcat/Km ratios, of the M214/247L and M229/247/317L mutantsincreased from 5.6 liters/(g · min) for the wild type to 8.1 and 7.5liters/(g · min), respectively, for the mutants. The specific activityof the M214/317L mutant increased slightly from 1.06 � 105

U/�mol before mutation to 1.18 � 105 U/�mol after mutation.The synergetic interaction of the five sites helps to improve thecatalytic efficiency of the enzyme.

Previously, it was reported that the oxidative stability of �-am-ylase can be improved by introducing mutations into Met residues(15, 18). For example, the replacement of Met 208 in the Bacillussp. TS-23 �-amylase with leucine enhanced its resistance to H2O2

(18). When Met 197 of �-amylase from Geobacillus stearothermo-philus US100 was replaced with alanine, resistance to chemicaloxidation was enhanced (15). However, in most cases, the oxida-tive stability improved only at the expense of a decreased alkalinestability, thermal stability, catalytic efficiency, or specific activity(15, 18), and this greatly limited the potential for the applicationof the targeted enzymes. In this work, the mutation of Met 247into leucine led to a 7-fold increase in oxidative stability and im-proved the alkaline stability, thermal stability, catalytic efficiency,specific activity, and tolerance to detergents (washing powder) tovarious extents. Here the M247L mutant has great potential in thetextile and detergent industries. This engineering work also dem-onstrates a means of remodeling enzymes to improve their per-formances and fulfill industrial requirements.

ACKNOWLEDGMENTS

This work was financially supported by the 863 Program (grants2011AA100905 and 2012AA022202), the 973 Program (grants2012CB720802 and 2012CB720806), Priority Academic Program Devel-opment of Jiangsu Higher Education Institutions, and the 111 Project(grant 111-2-06).

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