Amylase from wheat (Triticum aestivum) seeds: Its purification, biochemical attributes and active site studies

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a-Amylase from wheat (Triticum aestivum) seeds: Its purification,biochemical attributes and active site studies

Kritika Singh, Arvind M. Kayastha

School of Biotechnology, Faculty of Science, Banaras Hindu University, Varanasi 221005, India

a r t i c l e i n f o

Article history:

Received 30 October 2013Received in revised form 28 March 2014Accepted 11 April 2014Available online 23 April 2014

Keywords:

a-AmylaseGlycoproteinPurificationMS/MSSDSChemical modificationHistidine

a b s t r a c t

Glycosylateda-amylase from germinated wheat seeds (Triticum aestivum) has been purified to apparentelectrophoretic homogeneity with a final specific activity of 1372 U/mg. The enzyme preparation whenanalysed on SDSPAGE, displayed a single protein band with Mr33 kDa; Superdex 200 column showedMrof 32 kDa and MS/MS analysis further provided support for these values. The enzyme displayed itsoptimum catalytic activity at pH 5.0 and 68 C with an activation energy of 6.66 kcal/mol and Q101.42.The primary substrate for this hydrolase appears to be starch with Km 1.56 mg/mL, Vmax 1666.67 U/mgand kcat485 s

1 and hence is suitable for application in starch based industries. Thermal inactivationof a-amylase at 67C resulted in first-order kinetics with rate constant ( k) 0.0086 min1 and t1/280 min. The enzyme was susceptible to EDTA (10 mM) with irreversible loss of hydrolytic power. Inthe presence of 1.0 mM SDS, the enzyme lost only 14% and 23% activity in 24 and 48 h, respectively.Chemical modification studies showed that the enzyme contains histidine and carboxylic residues atits active site for its catalytic activity and possibly conserved areas.

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1. Introduction

a-Amylase, a metalloenzyme belonging to glycosyl hydrolase(GHH clan; GH-13 family), catalyses random endohydrolysis ofa-1,4 glycosidic bonds in starch and related carbohydrates withretention ofa-anomeric configuration in the products (Henrissat,1991; Kandra, Gyemant, Remenyik, Hovanszki, & Liptak, 2002).The repertoire of products upon amylolytic digestion ranges fromglucose and maltose to maltodextrins. The enzyme is ubiquitousin nature, extending to various realms of life from microorganisms,plants and animals as they play imperative role in carbohydratemetabolism. In plants, a-amylase expression materialises duringthe onset of seed germination and development and is promoted

in response to gibberellins and repressed by abscisic acid and sal-icylic acid (Xie, Zhang, Hanzlik, Cook, & Shen, 2007).There is a rich literature on the participation of sugar signal in

the mobilisation of endospermic starch during seed germination.While in the early stages of germination, sugar starvation conditionarises due to consumption of limited soluble sugars present in theseed (Perata, Guglielminetti, & Alpi, 1997), however, at a later stagethe plant hormone gibberellic acid emanates from the embryo andinitiatesde novosynthesis ofa-amylase in the aleurone layer and

its secretion into the starchy endosperm. The aleurone layerthereby undergoes apoptosis (Bethke, Lonsdale, Fath, & Jones,1999). The rapid escalation in endo-amylase level henceforthmobilises the storage starch and assists embryo to meet its nutri-tional requirements. Various aspects of this starch hydrolase havebeen investigated extensively: its protein structure and function(Kadziola et al.,1994; Machius, Wiegand, & Huber, 1995), its mech-anism of secretion through cell membrane and its industrial appli-cation (Saboury, 2002).

Structurally, a-amylase consists of a single polypeptide chainfolded into three domains (A, B and C). It has a characteristic(b/a)8barrel with conserved catalytic core (domain A), a protrusionbetween third strand and third helix of (b/a)8barrel having irreg-

ular b-like structure (domain B) and the C-terminal end of theamino acid sequence with key motif (domain C). Domain B isresponsible for the differences in substrate specificity of theenzyme and also for the stability of the enzyme (Svensson, 1994;Yang et al., 2013). On the other hand, domain C is thought to sta-bilise the catalytic site of the enzyme by shielding the hydrophobicpatch (Dauter et al., 1999; Yadav & Prakash, 2011). Calcium is piv-otal in the amylase structure requiring at least one calcium ion perenzyme molecule (Vallee, Stein, Sumerwell, & Fischer, 1959). Allknown a-amylases comprehend a structurally conserved calciumbinding site (Nielsen, Fuglsang, & Westh, 2003). Calcium not onlyoffers stability to a-amylase conformation but is also involved in

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Corresponding author. Tel.: +91 542 2368331; fax: +91 542 2368693.

E-mail address:[email protected](A.M. Kayastha).

Food Chemistry 162 (2014) 19

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substrate binding thus rendering stability to their higher orderedstructure by interacting with negatively charged residues such asaspartic acid and glutamic acid (Kumari, Singh, & Kayastha,2012). Its presence is quite indispensable and enhances enzymesthermostability.

Thermal stability is a desirable feature of the enzyme for itseconomic viability and longer shelf-life in various commercial pro-cesses. This attribute earns

a-amylase greater utility in terms of

competence and opens up several avenues in the industrial sector(Maarel & Veen, 2002). In terms of amylolytic action, starch hydrol-ysates are exploited for producing High Fructose Corn Syrup whichis a principal sweetener in processed foods and beverages.a-Amy-lase is utilised in varied commercial processes such as sugar syrup,brewing, ethanol production, textiles, paper and detergent indus-try, as an anti-staling agent in bread and bakery industry and insynthetic chemistry for the production of oligosaccharides(Kandra et al., 2002). They can be exploited for the developmentand designing of therapeutic agents against type II diabetes, obes-ity, hyperlipidemia and caries (Kandra et al., 2002).

However, the structural stability ofa-amylase can be jeopar-dised at the cost of extreme operating conditions prevalent inindustries especially with respect to pH and temperature. Theuse of aqueous sugar solvents and polyhydric alcohols are able toprotect the native structure of enzyme by altering its physico-chemical properties. The stabilising effect accounts from the posi-tive changes in the property of the proteins that are induced by theaddition of these substance (Arakawa & Timasheff, 1982; Yadav &Prakash, 2011).

Since cereal a-amylases have gained importance due to theirsuitability for biotechnological applications (Muralikrishna &Nirmala, 2005), and in the past there have been very few reportson purification ofa-amylase from plant sources, this paper inves-tigates the purification ofa-amylase and provides an insight intoits physico-chemical characterisation and recognition of aminoacid residues present at the active site.

2. Materials and methods

2.1. Plant materials and chemicals

Dry seeds of wheat (PBW 373 variety), purchased from a localmarket, were surface sterilised with 1% (v/v) sodium hypochloriteand thoroughly washed with Milli Q water. Germination of810 h imbibed seeds was effected in dark at 27C over moist filterpapers for about 36 h. Germinatedseeds were stored at20 C untiluse. All the chemicals for buffers were of analytical/electrophoreticgrade. Unless stated otherwise, all chemicals were purchased fromSigma (USA). Milli Q (Millipore, Bedford, MA, USA) water with aresistance of 18.2 MX cm was used all throughout the experiments.

2.2. Enzyme and protein assay

The hydrolytic activity of a-amylase was routinely assayedusing starch as substrate as described by Tripathi, Leggio,Mansfeld, Hofmann, and Kayastha (2007). Starch (potato starch,1%; w/v) solution, 0.5 mL, 0.3 mL sodium acetate buffer (100 mM,pH 5.0) and 0.1 mL double distilled water was incubated at 68 Cin a water bath (Multitemp; Pharmacia, Sweden) for 10 min. Theenzymic reaction was started by addition of 0.1 mL of suitablydiluted enzyme. The reaction was stopped after 5 min by adding0.5 mL 1 N HCl followed by rapid cooling. To 0.2 mL reaction mix-ture was added 0.1 mL 1 N HCl and 0.1 mL iodine solution. The

final volume was made to 15 mL and absorbance was observedat 610 nm. One unit of a-amylase was defined as amount of

enzyme, which caused a decrease of absorbance by 0.05 in starchiodine colour, under assay conditions.

The purified a-amylase activity was also assayed by using achromogenic substrate, starch azure. For this, starch azure suspen-sion (4.5 mL of 2%; w/v) was prepared in 20 mM sodium phosphatebuffer (pH 7.0) and was incubated at 37 C in a water bath. Thereaction was started by adding 0.5 mL (suitably diluted) of enzymeand incubated for 15 min. The reaction was stopped by adding of1 mL, 1 mol/L acetic acid. a-Amylase hydrolyses starch azure,releasing soluble dye that can be easily detected after filtrationof the reaction mixture using Whatman No.1 filter paper. Theabsorbance was measured at 595 nm. This was a qualitative confir-mation as subjected to the random cleavage property ofa-amylase,the oligodextrins produced on starch azure hydrolysis solubilisesthe dye, which now comes into the solution. The characteristic bluecolour produced during the hydrolysis reaction is direct evidenceto the presence ofa-amylase in the reaction mixture.

Protein assay was done according to the method of Lowry,Rosebrough, Farr, and Randall (1951)using crystalline BSA as stan-dard protein.

2.3. Purification ofa-amylase

All purification steps were executed at 4 C and centrifugationwas performed at 10,000 rpm for 15 min, unless stated otherwise.Column chromatographic flow rate was controlled and monitoredwith Microperpex peristaltic pump (Pharmacia, Sweden). Buffersused in each step included additives [1 mM dithiothreitol (DTT),5 mM CaCl2 and 0.5 mM phenylmethanesulfonylfluoride (PMSF)]and the final preparation contained a cocktail of commercial plantprotease inhibitors (Calbiochem, La Jolla).

2.3.1. Extraction

Purification in a typical batch included homogenisation of 50 g36-h germinated seeds dispersed in 150 mL chilled extraction buf-fer (50 mM sodium acetate buffer, pH 5.5) using a laboratory blen-

der followed by squeezing the homogenate through two layeredpre-washed muslin cloth and collected the supernatant bycentrifugation.

2.3.2. Ammonium sulphate fractionation

Proteins precipitating in the range 3065% ammonium sulphatesaturation were collected by adding small portions of salt and thenstirred for 2.5 h for the proteins to precipitate at 4 C. This was thencentrifuged and the pellet was collected and dissolved in minimumvolume of 50 mM sodium acetate buffer pH, 5.5.

2.3.3. Heat treatment

The above ammonium sulphate fraction was heated at 70 1 Cin a water bath for 10 min followed by centrifugation, which sep-

arated denaturedb-amylase. The remaining solution was then dia-lysed extensively against 50 mM sodium acetate buffer, pH 5.5 inorder to remove ammonium sulphate.

2.3.4. Epoxy activated Sepharose 6B affinity chromatography

Affinity chromatographic material was prepared as per themethod of Vretblad (Vretblad, 1974). Following dialysis, the super-natant (40 mg) was loaded onto affinity column (4.5 cm 3 cm)that was pre-equilibrated with 50 mM sodium acetate buffer (pH6.0) containing 5 mM CaCl2. The column was then washed with20 mM sodium acetate buffer (pH 6.0) containing 25 mM CaCl2and 200 mM NaCl until unbound proteins had been completelywashed off. Following this, the elution was carried out usingb-cyclodextrin (10 mg/mL) in washing buffer (Koshiba &

Minamikawa, 1981) at flow rate of 0.3 mL/min. The fractions(3 mL each) exhibiting high activity were pooled, dialysed against

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50 mM sodium acetate buffer (pH 6.0, containing 5 mM CaCl 2) andconcentrated using aquacide, polyethylene glycol (20 kDa) at 4 C.

2.3.5. Superdex 200 gel filtration on FPLC

Concentrated enzyme from the above step was loaded ontoSuperdex 200 column, pre-equilibrated with 50 mM sodium ace-tate (pH 5.5). The elution was done with the same buffer, contain-ing 200 mM NaCl at a flow rate of 0.5 mL/min. The active fractions(1 mL/fraction) were pooled, concentrated and stored with addedplant proteases inhibitor cocktail at 4 C.

2.4. Determination of molecular mass

SDSPAGE (2 cm, 5% stacking gel, pH 6.8 and 13 cm, 10% resolv-ing gel, pH 8.8) was prepared as described by Laemmli (1970)using a vertical gel electrophoresis apparatus (Monokin, India).An apparent subunit molecular mass of enzyme subunits were cal-culated by Alpha Innotech (AlphaImage, USA) from the dataobtained by SDSPAGE using protein marker with molecular massranging from 29 to 205 kDa. SDS gels were stained with CoomassieBrilliant Blue R.

2.5. Native PAGE and activity staining

Native PAGE (2 cm, 5% stacking gel, pH 6.8 and 13 cm, 8%resolving gel, pH 8.8) was carried out at 4 C and at a constant cur-rent supply of 5 mA until samples were stacked and then the cur-rent was increased to 10 mA. One half of the native PAGE gel wasstained with Coomassie brilliant blue R and the other half was usedfor activity staining according to protocol byKumari, Singh, Fitter,Polen, and Kayastha (2010). The gel was incubated at room tem-perature for 15 min in soluble starch (1%) prepared in 0.1 Msodium acetate buffer (pH 5.0), with constant stirring and thenstained with potassium iodide solution for visualising a-amylaseband.

2.6. Native molecular mass determination

The native mass of the purified protein was determined with gelfiltration using Superdex 200 on AKTA FPLC (GE Healthcare, Upp-sala) pre-equilibrated with 50 mM sodium acetate buffer pH 5.6containing 200 mM NaCl. The purified enzyme 400 lL was chro-matographed at a flow rate of 0.5 mL/min. Fractions (1 mL) werecollected and assayed fora-amylase activity. The native molecularmass was calculated from a plot ofVe/Vo against log of molecularmass using the following protein standards: Thyroglobulin(669 kDa), Aldolase (158 kDa), Conalbumin (75 kDa), Albumin(45 kDa) and Chymotrypsinogen A (25 kDa). The void volumewas determined using Blue dextran.

2.7. Glycoprotein properties

To test for the glycoprotein nature of the enzyme, Schiffsreagent was used. The enzyme sample (200 lL) was incubatedwith 100 lL of 10 mM sodium meta periodate for 10 min, prevent-ing its exposure to air, followed by addition of 300 lL of 0.5%Schiffs reagent to it. The mixture was incubated for 1 h and theabsorbance read at 550 nm.

2.8. Isoelectric focussing

In order to determine the isoelectric point of the enzymesample, a recent protocol was followed (Singh & Kayastha, 2012).Isoelectric focusing was carried out using immobiline dry strip

pH 310, 11 cm (GE healthcare, Uppsala). IPG buffer (pH range;310, GE healthcare) and bromophenol blue was added to the

purified enzyme. It was then loaded onto the strip and isoelectricfocusing was carried out using Ettan GE system for 16 h; thereafterthe strip was Coomassie stained to observe the protein band.

2.9. Mass spectrometry and database search

The above gel band was tryptic digested (using Sigma TrypsinProfile Kit) and submitted for mass spectrometry (Brker Daltonik,Bremen). The gel band of interest was excised from the Coomassiestained SDSPAGE gel and placed in a siliconised Eppendorf tube.The gel piece was destained using 200 mM ammonium bicarbon-ate and 40% acetonitrile. Next, the destained gel piece was driedand digested overnight with trypsin enzyme (0.4 lg). The superna-tant was then vacuum dried and resuspended in 0.1% trifluoroace-tic acid and 30% acetonitrile. Peptides were deposited on a matrixassisted laser desorption/ionisation time of flight (MALDI-TOF)plate, and over layered with a-cyano-4-hydroxy cinnamic acidmaldi matrix (10 mg/mL) in 70% acetonitrile with 0.1% trifluoro-acetic acid. MALDI analysis was performed in a linear positiveion mode.

The mass spectra were subjected to sequence database searchusing MS-Fit (Protein Prospector) against NCBInr plant databases.The spectra were analysed by MASCOT sequence matching soft-ware (www.matrixscience.com). Protein identifications were eval-uated on the basis of multiple variables such as score, no. ofpeptides matched, quality of the peptide maps besides similarityof experimental and theoretical protein molecular mass.

2.10. Kinetic studies

For all kinetic studies, the enzyme obtained after size-exclusionchromatography was utilised. The pH optimum was determined byassaying at different pH using various buffers viz., 100 mM sodiumacetate (4.05.6), 100 mM phosphate buffer (5.78.0) and 100 mMTris buffer (8.09.0). The optimum temperature was evaluated byassaying enzyme activity at temperatures ranging from 25 C to

85 C (1 C) in a water bath. The activation energy (Ea) was com-puted from the slope of the curve using Arrhenius plot. Thermalinactivation studies were performed by incubating enzyme at var-ious temperatures for different time intervals, followed by residualactivity assay under standard conditions. In order to determine theMichaelisMenten constant (Km), the enzyme was assayed in thepresence of varying concentration of starch (0.510 mg/mL) understandard assay conditions.

Different concentrations of additives (DTT, PMSF), salts (CaCl2,MgCl2, NaCl, KCl, HgCl2, AgCl, ZnCl2) and detergent (SDS) wereadded to the enzyme sample and the residual activity was mea-sured after 24 h. To study the effect of EDTA, purified amylasewas dialyzed against 50 mM sodium acetate buffer (pH 5.5) con-taining 10 mM EDTA at room temperature with hourly buffer

change. Loss of activity was monitored and reconstituted againstthe same buffer containing 50 mM of different metal ions (Ca2+,Na+, Mg2+) and the relative activity was quantified. All parameterswere the mean of duplicate determinations from two differentpreparations. The enzyme was tested for its ability to hydrolysevarious substrates. The activities against starch, amylase (potato),amylopectin (potato), dextrin III (corn), dextrin IV (potato), glyco-gen (oyster), maltose and pullulan (Aureobasidium pullulans) wereassayed using Bernfeld method (1955). Except for pullulan, glyco-gen and maltose, the buffer was slightly warmed to dissolve therest of the substrates.

2.11. Determination of pKa

The effect of pH on Vmax and Kmwas examined by determiningthea-amylase activity in 100 mM buffers of different pH (48).

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2.11.1. Inactivation ofa-amylase with 1-ethyl-3-[3-(dimethylamino)p

ropyl]carbodiimide (EDC)

Chemical modification ofa-amylase carboxylic acid side chainwith EDC was carried out in 100 mM phosphate buffer (pH 6.0 con-taining 0.2 M glycine ethyl ester). Enzyme (0.11 mg/mL) was incu-bated with desired amount of EDC with aliquots withdrawn atdifferent time intervals and assayed fora-amylase residual activ-ity. For protection studies, enzyme was incubated with EDC(0.1 mM) in presence of starch (5 mg/mL). Aliquots withdrawn atvarious time intervals were assayed for residual enzyme activity.

Experiments to rule out the presence of SH and tyrosine groupsat the enzymes active site were done by incubating EDC modifiedenzyme sample with N-ethyl maleimide (NEM; 2 mM) and withhydroxylamine (0.5 M), respectively.

2.11.2. Inactivation of a-amylase with diethylpyrocarbonate (DEP)

The method described by Srivastava and Kayastha (2001)wasused for histidine residue modification. DEP was diluted with eth-anol immediately before use. DEP concentration was measured byreacting an aliquot with 10 mM imidazole (pH 6.8) and monitoringthe absorbance at 230 nm. The extinction coefficient of imidazolewas 3 103 M1 cm1 (Miles, 1977). The enzyme solution(0.107 mg/mL) was incubated with DEP (0.5 mM final concentra-tion) in 0.1 M phosphate buffer (pH 6.8) at 30 C. The reactionwas monitored at 242 nm (De3.2 103 M1 cm1) in order to fol-low the progress of imidazole modification, whereas for kineticstudies aliquots withdrawn at regular time intervals were assayedfor residual a-amylase activity. Reactivation of DEP-inactivatedenzyme was prompted by incubating it with hydroxylamine(0.2 M, pH 7.0) and time dependent recovery was monitored at37 C. Protection against DEP-modification was checked by incu-bating enzyme with DEP in presence of starch (5 mg/mL) and theresidual enzyme activity was recorded at regular time intervals.

3. Results and discussion

3.1. Purification

a-Amylase from 36 h germinated Triticum aestivum was puri-fied with a final specific activity of about 1372 U/mg and an overallrecovery of 5% using an array of fractionation and chromatographictechniques as shown in Table 1A. The purification protocol wasreproducible every time we ran a batch of purification.a-Amylasepurified from barley showed specific activity of 1387 U/mg ( Bush,Sticher, Van Huysteee, Wagner, & Jones, 1989). Also, there havebeen few reports ofa-amylase purification from wheat with spe-cific activity of 800 U/mg (Sharma, Sharma, & Gupta, 2000),700 U/mg (Sharma et al., 2000) and 2244 U/mg (Machaiah &Vakil, 1984). In our experiments we concluded that an optimalextraction ofa-amylase could be achieved by using a ratio of 1:3(w/v) of germinated seeds to extraction buffer (50 mM sodium ace-

tate inclusive of 5 mM CaCl2, 1 mM DTT and 0.5 mM PMSF; pH 5.5)for preparing the homogenate (data not shown).

Crude extract so obtained was selectively fractionated withammonium sulphate in the range of 3065%. Following this, theheat treatment step provided efficiency to purification by eliminat-ing heat labile b-amylase (Kumari et al., 2010). Thereafter, affinitychromatography (Epoxy activated Sepharose 6B ligated withb-cyclodextrin) proved to be an imperative mode to purify a-amy-lase and this yielded a highly purified enzyme (Fig. 1A) with selec-tive elution by b-cyclodextrin (10 mg/mL) (Kumari et al., 2010;Tripathi et al., 2007). The enzyme was eluted as a single peak whenit was finally loaded onto Superdex 200 (Fig. 1B). This step showed

no further increment in specific activity thereby suggesting homo-geneity of the enzyme. In order to prevent proteolysis of theenzyme, 1% (v/v) protease inhibitor cocktail was added to the finalpreparation. Under this condition the enzyme was found to befairly stable showing 65% activity over 2 months of storage at 4 C.

Furthermore, the purified enzyme revealed a single proteinband corresponding to a molecular mass of 33 kDa on SDSPAGE(Fig. 2B). Native-PAGE profile displayed a protein band

Table 1A

Purification ofa-amylase from 36 h germinated wheat seeds (50 g).

Steps Total activity (Units) Total protein (mg) Specific activitya (Units mg1) Purification foldb Recovery %

Crude 28,728 636 45 1 100Ammonium sulphate (3065%) 27,900 207 135 3 97.1Heat treatment (70 C, 10 min) 16,236 79.2 205 5 56.5Epoxy activated Sepharose 6B 2987.34 2.47 1210 27 10.4Superdex 200 1426.5 1.04 1372 30 5

a Enzyme activity was determined using starch and protein determination was done using Lowry method Lowry (1951).b Fold purification was calculated with respect to the specific activity of the crude extract.

2 4 6 8 10 12 14

A280

0.0

0.1

0.2

0.3

0.4

0.5

A280

Activity (U/mL)

Fraction number

Activity(U/mL)

0

10

20

30

40

50

Fraction number

5 10 15 20 25 30

mA280

0

10

20

30

40

mA280

Activity (U/mL)

Activity(U/mL)

0

10

20

30

40

(A)

(B)

Fig. 1. (A) Elution profile for affinity chromatography ofa-amylase on b-cyclodex-

trin Sepharose 6B column. Elution was carried out with 10 mg/mLb-cyclodextrinand (B)a-amylase elution profile on Superdex 200.



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corresponding to activity staining (using Starch Iodine method)(Fig. 2B). The molecular mass as determined by Superdex 200size-exclusion chromatography was 32 kDa (Fig. 2A) whichmatches well with 33 kDa from SDSPAGE. These results clearlydemonstrate that the enzyme is monomeric in its native form.Low molecular weight a-amylase has been reported in pearl millet(31 kDa) (Muralikrishna & Nirmala, 2005) and Vigna mungo(43 kDa) (Koshiba & Minamikawa, 1981). The presence of a slightlydiffused protein bands under reducing and non-reducing condi-tions might be due to the glycosylated nature of the protein. A sin-gle band obtained on SDSPAGE was excised and after trypticdigestion was submitted for MS/MS analysis. The peptide mass fin-gerprint obtained (Fig. 2C) was matched with the available plantdatabase, which showed significant matches against a-amylasefrom Barley and Oryza sativawith a score of 570 and 156, respec-tively. The pI for the enzyme was found to be 8.0.

3.2. Biochemical properties

The effect of pH on wheata-amylase showed maximum activityat pH 5 (Fig. 3A). Temperature optimum was observed to be 68 C

(Fig. 3B), being indicative of a stable enzyme substrate complexformation, which protect enzyme from heat denaturation. Thereaction rate follows the Arrhenius equation which holds well inthe temperature range 2568C, with an activation energy

6.657 kcal/mol and Q10 of 1.42. The kinetics of starch hydrolysiswhen studied under standard experimental conditions, usingLineweaverBurk plot, revealed Km 1.56 mg/mL and Vmax1666.67 U/mg (Fig. 3C). Turnover number measuring the efficiencyof catalytic production of product under optimum conditions was485 s1 for starch. Thermal inactivation followed at 67 C, illus-trated first-order kinetics with t1/2 of 80 min and rate constant(k

) of 0.0086 min1 (Fig. 3D).Wheata-amylase was found to hydrolyse starch at a much fas-

ter rate than the other substrates studied (Table 1B). It furthershowed its inability to hydrolyse pullulan which has extensive a-1,6 glycosidic linkages and glycogen being more profuselybranched than starch, hence the rate of hydrolysis is quite lowwhereas other substrates have much higher rate of hydrolysisdue to presence ofa-1,4 glycosidic bonds in them. Oligosaccha-rides of glycoproteins are known to control their variety of biolog-ical and physiochemical properties relating to folding, secretion,solubility and stability (Kishore & Kayastha, 2012). Wheata-amy-lase showed magenta colour with Schiffs reagent indicating that itwas likely a glycosylated protein. Ethylene diamine tetra aceticacid (EDTA) had an irreversible effect on the a-amylase activity.On dialysing the wheata-amylase against 5 mM EDTA, all activitywas lost. This referred to the loss of Ca2+ which got sequestered byhexadentate ligand EDTA. Only 14% of enzymes activity could berecovered on addition of Ca2+. Also, substitution of Ca2+ with

Log Mr

1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8

ElutionVolume(m

L)

8

10

12

14

16

18

Thyroglobulin

(669 kDa)

Aldolase

(158 kDa)

Conalbumin(75 kDa)

Albumin (45 kDa)

mylase

Chymotrypsinogen(25 kDa)

(A)

(C)

(B)

Fig. 2. (A) Semi-logarithmic plot of elution volume against molecular mass; (B) electrophoresis pattern of purified wheat a-amylase. Lanes a and b show Coomassie BrilliantBlue R stained molecular weight markers and purified enzyme, respectively under reduced condition (SDSPAGE). Lane c shows Coomassie stained Native-PAGE under non-

reducing condition. Lane d shows activity staining of the enzyme using starch-iodine method and (C) peptide mass fingerprint spectra of the tryptic digest of a-amylase fromwheat after purification.

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Mg2+ and Na+ showed no improvement in the result. This indicatesthat Ca2+ is one of the important components for the enzymesstructure and function.

Recently, the mechanism of stabilisation upon calcium bindingof three homologous a-amylases was investigated by measuringthe unfolding kinetics (Kumari, Rosenkranz, Kayastha, & Fitter,2010). The enzyme activity was enhanced in the presence ofMg2+, Mn2+, Na+ and K+, whereas it showed complete inhibition

with Ag

+

, Hg

2+

and Zn

2+

. Enhancement of activity may be the resultfrom electrostatic stabilisation effect imparted by these alkali

metals. Zn2+ is known to inhibit a-amylase activity (Moranelli,Yaguchi, Calleja, & Nasim, 1987). Heavy metal ions inactivateenzymes non-competitively and this inhibition accounts from theirreaction with thiol groups or active site residues. Apart from thesethiols, the heavy metal ions are anticipated to react with the histi-dine groups and also the peptide linkages. The Ki obtained fromDixon plot for Ag+, Hg2+ and Zn2+ were 1.89 lM (Fig. 4A),0.702 lM(Fig. 4B) and 0.94 mM (Fig. 4C), respectively.

SDS, an anionic detergent, is significantly known to disrupt theproteins higher ordered structure. At 1 mM SDS concentration, theenzyme was functionally responsive and resisted denaturation asonly 14% and 23% of the activity was lost after 24 h and 48 h ofincubation, respectively. This could probably result from the gly-cosylated nature of wheat

a-amylase, leading to its poor binding

to SDS as has been shown recently for a-galactosidase from chick-peas (Singh & Kayastha, 2012).

3.3. Determination of pKa values

The effect of pH on the kinetic constants for wheata-amylase indifferent pH buffers illustrated values for pKa1 of 4.87 (Asp/Glu)and for pKa2 6.81 (His). These pKa values do not correspond tothe pKa of substrate starch (Potato starch, pKa 3.7). The effect ofpH on log(Vmax) is shown in Fig. 5A. Such apparent pKa valuesmay be perturbed due to the microenvironment created aroundamino acid groups, which influences their intrinsic pKas(Harris &Turner, 2002). Chemical modification studies were carried out to

probe the identity of amino acid residues and their respective rolesin catalytic function.

pH

3 4 5 6 7 8 9

Activit

y(UmL-1)

0

20

40

60

80

100

120

140

160

Sodium acetate

Sodium phosphate

Tris acetate

Temperature (C)

20 30 40 50 60 70 80 90

Activity(UmL-1)

20

40

60

80

100

120

140

1/[S]

.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2

1/v(moles/m

in/mg)-1

0.0000

0.0002

0.0004

0.0006

0.0008

0.0010

0.0012

0.0014

0.0016

0.0018

Time (min)

0 20 40 60 80 100 120

Log%ResidualAc

tivity

1.2

1.4

1.6

1.8

2.0

(A) (B)

(C) (D)

Fig. 3. (A)Effect of pH, (B)temperatureon theactivityof wheata-amylase, (C) LineweaverBurk plot for wheata-amylase for starch as substrate and (D) thermal inactivationof wheata-amylase at 67 C, showing first-order kinetics.

Table 1B

Relative substrate hydrolysis.

Substrate % Relative activity

Starch 100Amylopectin 27Amylose 18Maltose 17Dextrin III 15Dextrin IV 6.7Pullulan 3.0Glycogen 2.4

Activity assay was carried out by DNS method. 0.5 mL suitably diluted enzyme wasincubated with 0.5 mL 1% (w/v) substrate for 3 min and then 1 mL DNS reagent wasadded for colour development. The tube containing this reaction mixture was

incubated in a boiling water bath for 5 min and then cooled in running tap water.After addition of 10 mL of Milli Q water the absorbance was observed at 540 nm.One unit of activity was defined as the amount of enzyme required to produce1 lmol of reducing sugar.



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3.3.1. Inactivation with EDC

The presence of carboxyl groups at the active site ofa-amylaseis well-documented and thoroughly established (Kadziola, Abe,Svensson, & Haser, 1994; Machius et al., 1995) hence, modificationof wheat enzyme with EDC (0.1 mM) did show strong inhibition toenzyme activity with rate constant (kobs) of 0.126 min

1. Incuba-tion with the substrate resulted in the protection of the enzymeactivity with 82% of original activity, thus indicating the presenceof acidic group at the enzymes active site (Fig. 5B). EDC, besidesthe acidic group, can also modify tyrosine and SH group. However,when modified enzyme was incubated with hydroxylamine(0.5 M) for 48 h, it failed to retrieve the lost activity hence rulingout the possibility of tyrosine modification. Also, NEM an SH group

modifier, failed to inhibit a-amylase activity.

3.3.2. Inactivation with DEP

DEP readily reacts with imidazole groups and hence are knownto modify histidine residues with considerable specificity in pHrange (67). This reaction proceeds with an increase in absorbanceat 242 nm and the formation of an inactive ethoxyformylatedenzyme derivative. Wheat a-amylase modification with DEPresulting in enzymic inactivation was both time and modifier con-centration dependent (data not shown). The linearity of plot oflog% residual activity against time using linear regression analysisindicates that inactivation followed first-order kinetics with rateconstant (kobs) 0.112 min

1.Fig. 5C shows that a-amylase lost itsactivity in a single exponential decay when incubated with

0.5 mM DEP at pH 6.8 and 37C. Pre-incubation of enzyme withstarch for 5 min before DEP modification rendered significant

protection against the rate of inactivation with a retention of84.4% of original activity. This clearly suggested that the histidineresidue is located in the protein active site.

DEP, beside histidine can potentially modify lysine, tyrosine andsulfhydryl group. However, unlike histidine modification, the reac-tions with both lysine and sulfhydryl residues are irreversible.Hence, to unambiguously differentiate between histidine andnon-histidine modification, the reaction reversibility was checkedby treatment with hydroxylamine. Incubation of inactivatedenzyme (Residual activity 18%) with hydroxylamine regainednearly 65.4% of the original activity over a period of 3 h thus rulingout any modification of lysine or sulfhydryl group with disappear-ance of absorbance peak at 240 nm. The hydroxylamine treatment

on the ethoxyformylated histidine leads to opening of the imidaz-ole ring by Bamberger reaction resulting from the de-eth-oxyformylatin of histidine residues (Srivastava & Kayastha, 2001).Computation of a number of histidine groups modified by DEP atdifferent time intervals showed that at the initial stages the lossof activity was linearly related with moles of histidine residuesblocked (1:1) (Fig. 5D) but later on, this ratio increases thus indi-cating that the active site histidine is the most reactive, is modifiedfirst, and as its availability decreases, other histidine residues getmodified (Dua & Kochhar, 1985). On extrapolating the line, the %loss in activity against the modified histidine residues, it can beobserved that only one histidine is involved at the active site.

Referring to the accepted reaction mechanism of a-amylasecatalysis a-retaining double displacement, glutamic acid is

involved as acid/base catalyst and an aspartate as the nucleophileattacking the glucose ring (Maarel & Veen, 2002). Clearly, the

[Ag+](M)

0 5 10 15 20 25 30

1/v(m

oles/min/mg)-1

0.000

0.002

0.004

0.006

0.008

0.010

0.012

0.014

Starch 5 mg mL-1

Starch 10 mg mL-1

[Hg2+

](M)

0 1 2 3 4 5 6 7

1/v(m

oles/min/mg)-1

0.000

0.002

0.004

0.006

0.008

0.010

Starch 5 mg mL-1

Starch 10 mg mL-1

[Zn2+

](mM)

-1 0 1 2 3 4 5 6

1/v(moles/min/m

g)-1

0.000

0.001

0.002

0.003

0.004

0.005

0.006Starch 5 mg mL

-1

Starch 10 mg mL-1

(A) (B)

(C)

Fig. 4. Dixon plot for calculating Kifor (A) Ag+, (B) Hg2+ and (C) Zn2+.

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presence of histidine residue at the active site does not coincidewith its involvement in the mechanism but the loss of wheat a-amylase activity upon DEP treatment is quite conducive with itsrole in substrate hydrolysis. It has been observed that in a-amy-lase, the histidine residue is found conserved from microorganismsto mammals (Kumari et al., 2010), hence it is a reasonable specu-lation that during the course of evolution, histidine has acquireda unique role which is physiologically significant for the controlof starch digestion.

The present conclusion is further strengthened by site-directedmutagenesis studies which indicated that histidine modification,aligned within the catalytic site, drastically reduced the a-amy-lase hydrolytic activity (Sgaard, Kadziola, Haser, & Svensson,1993). Reports on its function as a transition state stabilizer dur-ing catalysis and that its replacement greatly diminishes the sta-bility of the transition state, suggest that its presence at the activesite being conserved and catalytic in nature (Bartlett, Porter,Borkakoti, & Thornton, 2002; Smith, Heschel, King, & Taubman,1999). Also, a comparative study of conserved regions and respec-tive catalytic active residues of a-amylases from Glycine max,AMY1 and AMY2 from Hordeum vulgare, Bacillus licheniformis(BLA), and Aspergillus oryzae (TAKA) was carried out using theClustalW for multiple sequence alignment. This suggested thatHis181 in soybean, His93 in AMY1, His92 in AMY2, His105 in BLA

and His122 in TAKA were conserved (Kumari et al., 2010).

4. Conclusions

The purified wheat a-amylase was found to be stable enzymeover 2 months at 4C. Having a high kcat, this demonstrates its abil-ity to hydrolyse starch efficiently. Its thermal stability further indi-cates its potential for industrial applications, especially for SDS-based detergent industries.

Acknowledgements

One of us (K.S.) would like to acknowledge the financial assis-tance from Council of Scientific and Industrial Research (CSIR),New Delhi in the form of Junior and Senior research fellowshipsto carry out this work. We also wish to thank Prof. D. Dash, atthe Department of Biochemistry, Institute of Medical Sciences,Banaras Hindu University, for providing the necessary facilitiesfor the pI determination.

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Time (min)

0 5 10 15 20 25

Log%Residual

Activity

1.0

1.2

1.4

1.6

1.8

2.0

2.25 mg mL

-1starch + Enzyme 0.1 mM EDC

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