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α-Amylase from wheat (Triticum aestivum) seeds: Its purification,

biochemical attributes and active site studies

Article in Food Chemistry · November 2014

DOI: 10.1016/[Link].2014.04.043

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Kritika Singh Arvind M Kayastha

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 Food Chemistry 162 (2014) 1–9

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: [Link]/locate/foodchem

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 a b s t r a c t

Article history: Glycosylated a-amylase from germinated wheat seeds (Triticum aestivum) has been purified to apparent
Received 30 October 2013 electrophoretic homogeneity with a final specific activity of 1372 U/mg. The enzyme preparation when
Received in revised form 28 March 2014 analysed on SDS–PAGE, displayed a single protein band with Mr 33 kDa; Superdex 200 column showed
Accepted 11 April 2014
Mr of 32 kDa and MS/MS analysis further provided support for these values. The enzyme displayed its
Available online 23 April 2014
optimum catalytic activity at pH 5.0 and 68 °C with an activation energy of 6.66 kcal/mol and Q10 1.42.
The primary substrate for this hydrolase appears to be starch with Km 1.56 mg/mL, Vmax 1666.67 U/mg
Keywords:
and kcat 485 s1 and hence is suitable for application in starch based industries. Thermal inactivation
a-Amylase
Glycoprotein
of a-amylase at 67 °C resulted in first-order kinetics with rate constant (k) 0.0086 min1 and t1/2
Purification 80 min. The enzyme was susceptible to EDTA (10 mM) with irreversible loss of hydrolytic power. In
MS/MS the presence of 1.0 mM SDS, the enzyme lost only 14% and 23% activity in 24 and 48 h, respectively.
SDS Chemical modification studies showed that the enzyme contains histidine and carboxylic residues at
Chemical modification its active site for its catalytic activity and possibly conserved areas.
Histidine Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction its secretion into the starchy endosperm. The aleurone layer
thereby undergoes apoptosis (Bethke, Lonsdale, Fath, & Jones,
a-Amylase, a metalloenzyme belonging to glycosyl hydrolase 1999). The rapid escalation in endo-amylase level henceforth
(GH–H clan; GH-13 family), catalyses random endohydrolysis of mobilises the storage starch and assists embryo to meet its nutri-
a-1,4 glycosidic bonds in starch and related carbohydrates with tional requirements. Various aspects of this starch hydrolase have
retention of a-anomeric configuration in the products (Henrissat, been investigated extensively: its protein structure and function
1991; Kandra, Gyemant, Remenyik, Hovanszki, & Liptak, 2002). (Kadziola et al.,1994; Machius, Wiegand, & Huber, 1995), its mech-
The repertoire of products upon amylolytic digestion ranges from anism of secretion through cell membrane and its industrial appli-
glucose and maltose to maltodextrins. The enzyme is ubiquitous cation (Saboury, 2002).
in nature, extending to various realms of life from microorganisms, Structurally, a-amylase consists of a single polypeptide chain
plants and animals as they play imperative role in carbohydrate folded into three domains (A, B and C). It has a characteristic
metabolism. In plants, a-amylase expression materialises during (b/a)8 barrel with conserved catalytic core (domain A), a protrusion
the onset of seed germination and development and is promoted between third strand and third helix of (b/a)8 barrel having irreg-
in response to gibberellins and repressed by abscisic acid and sal- ular b-like structure (domain B) and the C-terminal end of the
icylic acid (Xie, Zhang, Hanzlik, Cook, & Shen, 2007). amino acid sequence with key motif (domain C). Domain B is
There is a rich literature on the participation of sugar signal in responsible for the differences in substrate specificity of the
the mobilisation of endospermic starch during seed germination. enzyme and also for the stability of the enzyme (Svensson, 1994;
While in the early stages of germination, sugar starvation condition Yang et al., 2013). On the other hand, domain C is thought to sta-
arises due to consumption of limited soluble sugars present in the bilise the catalytic site of the enzyme by shielding the hydrophobic
seed (Perata, Guglielminetti, & Alpi, 1997), however, at a later stage patch (Dauter et al., 1999; Yadav & Prakash, 2011). Calcium is piv-
the plant hormone gibberellic acid emanates from the embryo and otal in the amylase structure requiring at least one calcium ion per
initiates de novo synthesis of a-amylase in the aleurone layer and enzyme molecule (Vallee, Stein, Sumerwell, & Fischer, 1959). All
known a-amylases comprehend a structurally conserved calcium
binding site (Nielsen, Fuglsang, & Westh, 2003). Calcium not only
⇑ Corresponding author. Tel.: +91 542 2368331; fax: +91 542 2368693. offers stability to a-amylase conformation but is also involved in
E-mail address: kayasthabhu@[Link] (A.M. Kayastha).

[Link]
0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
2 K. Singh, A.M. Kayastha / Food Chemistry 162 (2014) 1–9

substrate binding thus rendering stability to their higher ordered enzyme, which caused a decrease of absorbance by 0.05 in starch
structure by interacting with negatively charged residues such as iodine colour, under assay conditions.
aspartic acid and glutamic acid (Kumari, Singh, & Kayastha, The purified a-amylase activity was also assayed by using a
2012). Its presence is quite indispensable and enhances enzyme’s chromogenic substrate, starch azure. For this, starch azure suspen-
thermostability. sion (4.5 mL of 2%; w/v) was prepared in 20 mM sodium phosphate
Thermal stability is a desirable feature of the enzyme for its buffer (pH 7.0) and was incubated at 37 °C in a water bath. The
economic viability and longer shelf-life in various commercial pro- reaction was started by adding 0.5 mL (suitably diluted) of enzyme
cesses. This attribute earns a-amylase greater utility in terms of and incubated for 15 min. The reaction was stopped by adding of
competence and opens up several avenues in the industrial sector 1 mL, 1 mol/L acetic acid. a-Amylase hydrolyses starch azure,
(Maarel & Veen, 2002). In terms of amylolytic action, starch hydrol- releasing soluble dye that can be easily detected after filtration
ysates are exploited for producing High Fructose Corn Syrup which of the reaction mixture using Whatman No.1 filter paper. The
is a principal sweetener in processed foods and beverages. a-Amy- absorbance was measured at 595 nm. This was a qualitative confir-
lase is utilised in varied commercial processes such as sugar syrup, mation as subjected to the random cleavage property of a-amylase,
brewing, ethanol production, textiles, paper and detergent indus- the oligodextrins produced on starch azure hydrolysis solubilises
try, as an anti-staling agent in bread and bakery industry and in the dye, which now comes into the solution. The characteristic blue
synthetic chemistry for the production of oligosaccharides colour produced during the hydrolysis reaction is direct evidence
(Kandra et al., 2002). They can be exploited for the development to the presence of a-amylase in the reaction mixture.
and designing of therapeutic agents against type II diabetes, obes- Protein assay was done according to the method of Lowry,
ity, hyperlipidemia and caries (Kandra et al., 2002). Rosebrough, Farr, and Randall (1951) using crystalline BSA as stan-
However, the structural stability of a-amylase can be jeopar- dard protein.
dised at the cost of extreme operating conditions prevalent in
industries especially with respect to pH and temperature. The 2.3. Purification of a-amylase
use of aqueous sugar solvents and polyhydric alcohols are able to
protect the native structure of enzyme by altering its physico- All purification steps were executed at 4 °C and centrifugation
chemical properties. The stabilising effect accounts from the posi- was performed at 10,000 rpm for 15 min, unless stated otherwise.
tive changes in the property of the proteins that are induced by the Column chromatographic flow rate was controlled and monitored
addition of these substance (Arakawa & Timasheff, 1982; Yadav & with Microperpex peristaltic pump (Pharmacia, Sweden). Buffers
Prakash, 2011). used in each step included additives [1 mM dithiothreitol (DTT),
Since cereal a-amylases have gained importance due to their 5 mM CaCl2 and 0.5 mM phenylmethanesulfonylfluoride (PMSF)]
suitability for biotechnological applications (Muralikrishna & and the final preparation contained a cocktail of commercial plant
Nirmala, 2005), and in the past there have been very few reports protease inhibitors (Calbiochem, La Jolla).
on purification of a-amylase from plant sources, this paper inves-
tigates the purification of a-amylase and provides an insight into 2.3.1. Extraction
its physico-chemical characterisation and recognition of amino Purification in a typical batch included homogenisation of 50 g
acid residues present at the active site. 36-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 layered
2. Materials and methods pre-washed muslin cloth and collected the supernatant by
centrifugation.
2.1. Plant materials and chemicals
2.3.2. Ammonium sulphate fractionation
Dry seeds of wheat (PBW 373 variety), purchased from a local Proteins precipitating in the range 30–65% ammonium sulphate
market, were surface sterilised with 1% (v/v) sodium hypochlorite saturation were collected by adding small portions of salt and then
and thoroughly washed with Milli Q water. Germination of stirred for 2.5 h for the proteins to precipitate at 4 °C. This was then
8–10 h imbibed seeds was effected in dark at 27 °C over moist filter centrifuged and the pellet was collected and dissolved in minimum
papers for about 36 h. Germinated seeds were stored at 20 °C until volume of 50 mM sodium acetate buffer pH, 5.5.
use. All the chemicals for buffers were of analytical/electrophoretic
grade. Unless stated otherwise, all chemicals were purchased from 2.3.3. Heat treatment
Sigma (USA). Milli Q (Millipore, Bedford, MA, USA) water with a The above ammonium sulphate fraction was heated at 70 ± 1 °C
resistance of 18.2 MX cm was used all throughout the experiments. in a water bath for 10 min followed by centrifugation, which sep-
arated denatured b-amylase. The remaining solution was then dia-
lysed extensively against 50 mM sodium acetate buffer, pH 5.5 in
2.2. Enzyme and protein assay order to remove ammonium sulphate.

The hydrolytic activity of a-amylase was routinely assayed 2.3.4. Epoxy activated Sepharose 6B affinity chromatography
using starch as substrate as described by Tripathi, Leggio, Affinity chromatographic material was prepared as per the
Mansfeld, Hofmann, and Kayastha (2007). Starch (potato starch, method of Vretblad (Vretblad, 1974). Following dialysis, the super-
1%; w/v) solution, 0.5 mL, 0.3 mL sodium acetate buffer (100 mM, natant (40 mg) was loaded onto affinity column (4.5 cm  3 cm)
pH 5.0) and 0.1 mL double distilled water was incubated at 68 °C that was pre-equilibrated with 50 mM sodium acetate buffer (pH
in a water bath (Multitemp; Pharmacia, Sweden) for 10 min. The 6.0) containing 5 mM CaCl2. The column was then washed with
enzymic reaction was started by addition of 0.1 mL of suitably 20 mM sodium acetate buffer (pH 6.0) containing 25 mM CaCl2
diluted enzyme. The reaction was stopped after 5 min by adding and 200 mM NaCl until unbound proteins had been completely
0.5 mL 1 N HCl followed by rapid cooling. To 0.2 mL reaction mix- washed off. Following this, the elution was carried out using
ture was added 0.1 mL 1 N HCl and 0.1 mL iodine solution. The b-cyclodextrin (10 mg/mL) in washing buffer (Koshiba &
final volume was made to 15 mL and absorbance was observed Minamikawa, 1981) at flow rate of 0.3 mL/min. The fractions
at 610 nm. One unit of a-amylase was defined as amount of (3 mL each) exhibiting high activity were pooled, dialysed against
 K. Singh, A.M. Kayastha / Food Chemistry 162 (2014) 1–9 3

50 mM sodium acetate buffer (pH 6.0, containing 5 mM CaCl2) and purified enzyme. It was then loaded onto the strip and isoelectric
concentrated using aquacide, polyethylene glycol (20 kDa) at 4 °C. focusing was carried out using Ettan GE system for 16 h; thereafter
the strip was Coomassie stained to observe the protein band.
2.3.5. Superdex 200 gel filtration on FPLC
Concentrated enzyme from the above step was loaded onto 2.9. Mass spectrometry and database search
Superdex 200 column, pre-equilibrated with 50 mM sodium ace-
tate (pH 5.5). The elution was done with the same buffer, contain- The above gel band was tryptic digested (using Sigma Trypsin
ing 200 mM NaCl at a flow rate of 0.5 mL/min. The active fractions Profile Kit) and submitted for mass spectrometry (Brüker Daltonik,
(1 mL/fraction) were pooled, concentrated and stored with added Bremen). The gel band of interest was excised from the Coomassie
plant proteases inhibitor cocktail at 4 °C. stained SDS–PAGE gel and placed in a siliconised Eppendorf tube.
The gel piece was destained using 200 mM ammonium bicarbon-
2.4. Determination of molecular mass ate and 40% acetonitrile. Next, the destained gel piece was dried
and digested overnight with trypsin enzyme (0.4 lg). The superna-
SDS–PAGE (2 cm, 5% stacking gel, pH 6.8 and 13 cm, 10% resolv- tant was then vacuum dried and resuspended in 0.1% trifluoroace-
ing gel, pH 8.8) was prepared as described by Laemmli (1970) tic acid and 30% acetonitrile. Peptides were deposited on a matrix
using a vertical gel electrophoresis apparatus (Monokin, India). assisted laser desorption/ionisation time of flight (MALDI-TOF)
An apparent subunit molecular mass of enzyme subunits were cal- plate, and over layered with a-cyano-4-hydroxy cinnamic acid
culated by Alpha Innotech (AlphaImage, USA) from the data maldi matrix (10 mg/mL) in 70% acetonitrile with 0.1% trifluoro-
obtained by SDS–PAGE using protein marker with molecular mass acetic acid. MALDI analysis was performed in a linear positive
ranging from 29 to 205 kDa. SDS gels were stained with Coomassie ion mode.
Brilliant Blue R. The mass spectra were subjected to sequence database search
using MS-Fit (Protein Prospector) against NCBInr plant databases.
2.5. Native PAGE and activity staining The spectra were analysed by MASCOT sequence matching soft-
ware ([Link]). Protein identifications were eval-
Native PAGE (2 cm, 5% stacking gel, pH 6.8 and 13 cm, 8% uated on the basis of multiple variables such as score, no. of
resolving gel, pH 8.8) was carried out at 4 °C and at a constant cur- peptides matched, quality of the peptide maps besides similarity
rent supply of 5 mA until samples were stacked and then the cur- of experimental and theoretical protein molecular mass.
rent was increased to 10 mA. One half of the native PAGE gel was
stained with Coomassie brilliant blue R and the other half was used 2.10. Kinetic studies
for activity staining according to protocol by Kumari, Singh, Fitter,
Polen, and Kayastha (2010). The gel was incubated at room tem- For all kinetic studies, the enzyme obtained after size-exclusion
perature for 15 min in soluble starch (1%) prepared in 0.1 M chromatography was utilised. The pH optimum was determined by
sodium acetate buffer (pH 5.0), with constant stirring and then assaying at different pH using various buffers viz., 100 mM sodium
stained with potassium iodide solution for visualising a-amylase acetate (4.0–5.6), 100 mM phosphate buffer (5.7–8.0) and 100 mM
band. Tris buffer (8.0–9.0). The optimum temperature was evaluated by
assaying enzyme activity at temperatures ranging from 25 °C to
2.6. Native molecular mass determination 85 °C (±1 °C) in a water bath. The activation energy (Ea) was com-
puted from the slope of the curve using Arrhenius plot. Thermal
The native mass of the purified protein was determined with gel inactivation studies were performed by incubating enzyme at var-
filtration using Superdex 200 on AKTA FPLC (GE Healthcare, Upp- ious temperatures for different time intervals, followed by residual
sala) pre-equilibrated with 50 mM sodium acetate buffer pH 5.6 activity assay under standard conditions. In order to determine the
containing 200 mM NaCl. The purified enzyme 400 lL was chro- Michaelis–Menten constant (Km), the enzyme was assayed in the
matographed at a flow rate of 0.5 mL/min. Fractions (1 mL) were presence of varying concentration of starch (0.5–10 mg/mL) under
collected and assayed for a-amylase activity. The native molecular standard assay conditions.
mass was calculated from a plot of Ve/Vo against log of molecular Different concentrations of additives (DTT, PMSF), salts (CaCl2,
mass using the following protein standards: Thyroglobulin MgCl2, NaCl, KCl, HgCl2, AgCl, ZnCl2) and detergent (SDS) were
(669 kDa), Aldolase (158 kDa), Conalbumin (75 kDa), Albumin added to the enzyme sample and the residual activity was mea-
(45 kDa) and Chymotrypsinogen A (25 kDa). The void volume sured after 24 h. To study the effect of EDTA, purified amylase
was determined using Blue dextran. was dialyzed against 50 mM sodium acetate buffer (pH 5.5) con-
taining 10 mM EDTA at room temperature with hourly buffer
2.7. Glycoprotein properties change. Loss of activity was monitored and reconstituted against
the same buffer containing 50 mM of different metal ions (Ca2+,
To test for the glycoprotein nature of the enzyme, Schiff’s Na+, Mg2+) and the relative activity was quantified. All parameters
reagent was used. The enzyme sample (200 lL) was incubated were the mean of duplicate determinations from two different
with 100 lL of 10 mM sodium meta periodate for 10 min, prevent- preparations. The enzyme was tested for its ability to hydrolyse
ing its exposure to air, followed by addition of 300 lL of 0.5% various substrates. The activities against starch, amylase (potato),
Schiff’s reagent to it. The mixture was incubated for 1 h and the amylopectin (potato), dextrin III (corn), dextrin IV (potato), glyco-
absorbance read at 550 nm. gen (oyster), maltose and pullulan (Aureobasidium pullulans) were
assayed using Bernfeld method (1955). Except for pullulan, glyco-
2.8. Isoelectric focussing gen and maltose, the buffer was slightly warmed to dissolve the
rest of the substrates.
In order to determine the isoelectric point of the enzyme
sample, a recent protocol was followed (Singh & Kayastha, 2012). 2.11. Determination of pKa
Isoelectric focusing was carried out using immobiline dry strip
pH 3–10, 11 cm (GE healthcare, Uppsala). IPG buffer (pH range; The effect of pH on Vmax and Km was examined by determining
3–10, GE healthcare) and bromophenol blue was added to the the a-amylase activity in 100 mM buffers of different pH (4–8).
4 K. Singh, A.M. Kayastha / Food Chemistry 162 (2014) 1–9

Table 1A
Purification of a-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 1 100
Ammonium sulphate (30–65%) 27,900 207 135 3 97.1
Heat treatment (70 °C, 10 min) 16,236 79.2 205 5 56.5
Epoxy activated Sepharose 6B 2987.34 2.47 1210 27 10.4
Superdex 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.

2.11.1. Inactivation of a-amylase with 1-ethyl-3-[3-(dimethylamino)p Crude extract so obtained was selectively fractionated with
ropyl]carbodiimide (EDC) ammonium sulphate in the range of 30–65%. Following this, the
Chemical modification of a-amylase carboxylic acid side chain heat treatment step provided efficiency to purification by eliminat-
with EDC was carried out in 100 mM phosphate buffer (pH 6.0 con- ing heat labile b-amylase (Kumari et al., 2010). Thereafter, affinity
taining 0.2 M glycine ethyl ester). Enzyme (0.11 mg/mL) was incu- chromatography (Epoxy activated Sepharose 6B ligated with
bated with desired amount of EDC with aliquots withdrawn at b-cyclodextrin) proved to be an imperative mode to purify a-amy-
different time intervals and assayed for a-amylase residual activ- lase and this yielded a highly purified enzyme (Fig. 1A) with selec-
ity. For protection studies, enzyme was incubated with EDC tive elution by b-cyclodextrin (10 mg/mL) (Kumari et al., 2010;
(0.1 mM) in presence of starch (5 mg/mL). Aliquots withdrawn at Tripathi et al., 2007). The enzyme was eluted as a single peak when
various time intervals were assayed for residual enzyme activity. it was finally loaded onto Superdex 200 (Fig. 1B). This step showed
Experiments to rule out the presence of SH and tyrosine groups no further increment in specific activity thereby suggesting homo-
at the enzyme’s active site were done by incubating EDC modified geneity of the enzyme. In order to prevent proteolysis of the
enzyme sample with N-ethyl maleimide (NEM; 2 mM) and with enzyme, 1% (v/v) protease inhibitor cocktail was added to the final
hydroxylamine (0.5 M), respectively. preparation. Under this condition the enzyme was found to be
fairly stable showing 65% activity over 2 months of storage at 4 °C.
Furthermore, the purified enzyme revealed a single protein
2.11.2. Inactivation of a-amylase with diethylpyrocarbonate (DEP)
band corresponding to a molecular mass of 33 kDa on SDS–PAGE
The method described by Srivastava and Kayastha (2001) was
(Fig. 2B). Native-PAGE profile displayed a protein band
used for histidine residue modification. DEP was diluted with eth-
anol immediately before use. DEP concentration was measured by
reacting an aliquot with 10 mM imidazole (pH 6.8) and monitoring
the absorbance at 230 nm. The extinction coefficient of imidazole
(A) 0.5 50

was 3  103 M1 cm1 (Miles, 1977). The enzyme solution

A280
Activity (U/mL) 40
0.4
(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 reaction
30
was monitored at 242 nm (De 3.2  103 M1 cm1) in order to fol- 0.3

Activity (U/mL)
low the progress of imidazole modification, whereas for kinetic
A280

20
studies aliquots withdrawn at regular time intervals were assayed 0.2
for residual a-amylase activity. Reactivation of DEP-inactivated
enzyme was prompted by incubating it with hydroxylamine 0.1
10

(0.2 M, pH 7.0) and time dependent recovery was monitored at

37 °C. Protection against DEP-modification was checked by incu- 0.0 0
bating enzyme with DEP in presence of starch (5 mg/mL) and the
residual enzyme activity was recorded at regular time intervals.
2 4 6 8 10 12 14
Fraction number
3. Results and discussion
(B) 40 40

mA280
3.1. Purification
Activity (U/mL)
30 30

a-Amylase from 36 h germinated Triticum aestivum was puri-

fied with a final specific activity of about 1372 U/mg and an overall
Activity (U/mL)

20
recovery of 5% using an array of fractionation and chromatographic 20
mA280

techniques as shown in Table 1A. The purification protocol was

reproducible every time we ran a batch of purification. a-Amylase 10 10
purified from barley showed specific activity of 1387 U/mg (Bush,
Sticher, Van Huysteee, Wagner, & Jones, 1989). Also, there have
been few reports of a-amylase purification from wheat with spe- 0 0

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 optimal 5 10 15 20 25 30

extraction of a-amylase could be achieved by using a ratio of 1:3 Fraction number

(w/v) of germinated seeds to extraction buffer (50 mM sodium ace- Fig. 1. (A) Elution profile for affinity chromatography of a-amylase on b-cyclodex-
tate inclusive of 5 mM CaCl2, 1 mM DTT and 0.5 mM PMSF; pH 5.5) trin Sepharose 6B column. Elution was carried out with 10 mg/mL b-cyclodextrin
for preparing the homogenate (data not shown). and (B) a-amylase elution profile on Superdex 200.
 K. Singh, A.M. Kayastha / Food Chemistry 162 (2014) 1–9 5

corresponding to activity staining (using Starch Iodine method) 6.657 kcal/mol and Q10 of 1.42. The kinetics of starch hydrolysis
(Fig. 2B). The molecular mass as determined by Superdex 200 when studied under standard experimental conditions, using
size-exclusion chromatography was 32 kDa (Fig. 2A) which Lineweaver–Burk plot, revealed Km 1.56 mg/mL and Vmax
matches well with 33 kDa from SDS–PAGE. These results clearly 1666.67 U/mg (Fig. 3C). Turnover number measuring the efficiency
demonstrate that the enzyme is monomeric in its native form. of catalytic production of product under optimum conditions was
Low molecular weight a-amylase has been reported in pearl millet 485 s1 for starch. Thermal inactivation followed at 67 °C, illus-
(31 kDa) (Muralikrishna & Nirmala, 2005) and Vigna mungo trated first-order kinetics with t1/2 of 80 min and rate constant
(43 kDa) (Koshiba & Minamikawa, 1981). The presence of a slightly (k) of 0.0086 min1 (Fig. 3D).
diffused protein bands under reducing and non-reducing condi- Wheat a-amylase was found to hydrolyse starch at a much fas-
tions might be due to the glycosylated nature of the protein. A sin- ter rate than the other substrates studied (Table 1B). It further
gle band obtained on SDS–PAGE was excised and after tryptic showed its inability to hydrolyse pullulan which has extensive a-
digestion was submitted for MS/MS analysis. The peptide mass fin- 1,6 glycosidic linkages and glycogen being more profusely
gerprint obtained (Fig. 2C) was matched with the available plant branched than starch, hence the rate of hydrolysis is quite low
database, which showed significant matches against a-amylase whereas other substrates have much higher rate of hydrolysis
from Barley and Oryza sativa with a score of 570 and 156, respec- due to presence of a-1,4 glycosidic bonds in them. Oligosaccha-
tively. The pI for the enzyme was found to be 8.0. 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). Wheat a-amy-
3.2. Biochemical properties lase showed magenta colour with Schiff’s reagent indicating that it
was likely a glycosylated protein. Ethylene diamine tetra acetic
The effect of pH on wheat a-amylase showed maximum activity acid (EDTA) had an irreversible effect on the a-amylase activity.
at pH 5 (Fig. 3A). Temperature optimum was observed to be 68 °C On dialysing the wheat a-amylase against 5 mM EDTA, all activity
(Fig. 3B), being indicative of a stable enzyme substrate complex was lost. This referred to the loss of Ca2+ which got sequestered by
formation, which protect enzyme from heat denaturation. The hexadentate ligand EDTA. Only 14% of enzyme’s activity could be
reaction rate follows the Arrhenius equation which holds well in recovered on addition of Ca2+. Also, substitution of Ca2+ with
the temperature range 25–68 °C, with an activation energy

(A) (B)
18 Chymotrypsinogen
(25 kDa)

α−Α
Αmylase
16 Albumin
(45 kDa)
Elution Volume (mL)

Conalbumin
14 (75 kDa)

Aldolase
(158 kDa)

12

10

Thyroglobulin
(669 kDa)
8
1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8

Log Mr

(C)

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 Brilliant
Blue R stained molecular weight markers and purified enzyme, respectively under reduced condition (SDS–PAGE). 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 from
wheat after purification.
6 K. Singh, A.M. Kayastha / Food Chemistry 162 (2014) 1–9

(A) 160
Sodium acetate
(B) 140

140 Sodium phosphate

Tris acetate 120
120

100
Activity (U mL-1)

Activity (U mL-1)
100

80 80

60
60
40

40
20

0 20
3 4 5 6 7 8 9 20 30 40 50 60 70 80 90

pH Temperature (°C)

(C) 0.0018
(D)
0.0016 2.0
1/v (μmoles/min/mg)-1

0.0014

Log % Residual Activity

1.8
0.0012

0.0010
1.6
0.0008

0.0006
1.4
0.0004

0.0002 1.2

0.0000
0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 0 20 40 60 80 100 120

1/[S] Time (min)

Fig. 3. (A) Effect of pH, (B) temperature on the activity of wheat a-amylase, (C) Lineweaver–Burk plot for wheat a-amylase for starch as substrate and (D) thermal inactivation
of wheat a-amylase at 67 °C, showing first-order kinetics.

Table 1B metals. Zn2+ is known to inhibit a-amylase activity (Moranelli,

Relative substrate hydrolysis. Yaguchi, Calleja, & Nasim, 1987). Heavy metal ions inactivate
enzymes non-competitively and this inhibition accounts from their
Substrate % Relative activity
reaction with thiol groups or active site residues. Apart from these
Starch 100
thiols, the heavy metal ions are anticipated to react with the histi-
Amylopectin 27
Amylose 18 dine groups and also the peptide linkages. The Ki obtained from
Maltose 17 Dixon plot for Ag+, Hg2+ and Zn2+ were 1.89 lM (Fig. 4A),
Dextrin III 15 0.702 lM (Fig. 4B) and 0.94 mM (Fig. 4C), respectively.
Dextrin IV 6.7 SDS, an anionic detergent, is significantly known to disrupt the
Pullulan 3.0
Glycogen 2.4
proteins’ higher ordered structure. At 1 mM SDS concentration, the
enzyme was functionally responsive and resisted denaturation as
Activity assay was carried out by DNS method. 0.5 mL suitably diluted enzyme was only 14% and 23% of the activity was lost after 24 h and 48 h of
incubated with 0.5 mL 1% (w/v) substrate for 3 min and then 1 mL DNS reagent was
added for colour development. The tube containing this reaction mixture was
incubation, respectively. This could probably result from the gly-
incubated in a boiling water bath for 5 min and then cooled in running tap water. cosylated nature of wheat a-amylase, leading to its poor binding
After addition of 10 mL of Milli Q water the absorbance was observed at 540 nm. to SDS as has been shown recently for a-galactosidase from chick-
One unit of activity was defined as the amount of enzyme required to produce peas (Singh & Kayastha, 2012).
1 lmol of reducing sugar.

3.3. Determination of pKa values

Mg2+ and Na+ showed no improvement in the result. This indicates The effect of pH on the kinetic constants for wheat a-amylase in
that Ca2+ is one of the important components for the enzyme’s different pH buffers illustrated values for pKa1 of 4.87 (Asp/Glu)
structure and function. and for pKa2 6.81 (His). These pKa values do not correspond to
Recently, the mechanism of stabilisation upon calcium binding the pKa of substrate starch (Potato starch, pKa 3.7). The effect of
of three homologous a-amylases was investigated by measuring pH on log(Vmax) is shown in Fig. 5A. Such apparent pKa values
the unfolding kinetics (Kumari, Rosenkranz, Kayastha, & Fitter, may be perturbed due to the microenvironment created around
2010). The enzyme activity was enhanced in the presence of amino acid groups, which influences their intrinsic pKas (Harris &
Mg2+, Mn2+, Na+ and K+, whereas it showed complete inhibition Turner, 2002). Chemical modification studies were carried out to
with Ag+, Hg2+ and Zn2+. Enhancement of activity may be the result probe the identity of amino acid residues and their respective roles
from electrostatic stabilisation effect imparted by these alkali in catalytic function.
 K. Singh, A.M. Kayastha / Food Chemistry 162 (2014) 1–9 7

(A) 0.014
Starch 5 mg mL-1
(B) 0.010

Starch 10 mg mL-1 Starch 5 mg mL-1

0.012 Starch 10 mg mL-1
0.008
1/v (μmoles/min/mg)-1

1/v (μmoles/min/mg)-1
0.010

0.006
0.008

0.006
0.004

0.004

0.002
0.002

0.000 0.000
0 5 10 15 20 25 30 0 1 2 3 4 5 6 7

+ 2+
[Ag ](μM) [Hg ](μM)

(C) 0.006
Starch 5 mg mL-1
Starch 10 mg mL-1
0.005
1/v (μmoles/min/mg)-1

0.004

0.003

0.002

0.001

0.000
-1 0 1 2 3 4 5 6

[Zn2+](mM)

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

3.3.1. Inactivation with EDC protection against the rate of inactivation with a retention of
The presence of carboxyl groups at the active site of a-amylase 84.4% of original activity. This clearly suggested that the histidine
is well-documented and thoroughly established (Kadziola, Abe, residue is located in the protein active site.
Svensson, & Haser, 1994; Machius et al., 1995) hence, modification DEP, beside histidine can potentially modify lysine, tyrosine and
of wheat enzyme with EDC (0.1 mM) did show strong inhibition to sulfhydryl group. However, unlike histidine modification, the reac-
enzyme activity with rate constant (kobs) of 0.126 min1. Incuba- tions with both lysine and sulfhydryl residues are irreversible.
tion with the substrate resulted in the protection of the enzyme Hence, to unambiguously differentiate between histidine and
activity with 82% of original activity, thus indicating the presence non-histidine modification, the reaction reversibility was checked
of acidic group at the enzyme’s active site (Fig. 5B). EDC, besides by treatment with hydroxylamine. Incubation of inactivated
the acidic group, can also modify tyrosine and SH group. However, enzyme (Residual activity ’ 18%) with hydroxylamine regained
when modified enzyme was incubated with hydroxylamine nearly 65.4% of the original activity over a period of 3 h thus ruling
(0.5 M) for 48 h, it failed to retrieve the lost activity hence ruling out any modification of lysine or sulfhydryl group with disappear-
out the possibility of tyrosine modification. Also, NEM an SH group ance of absorbance peak at 240 nm. The hydroxylamine treatment
modifier, failed to inhibit a-amylase activity. on the ethoxyformylated histidine leads to opening of the imidaz-
ole ring by Bamberger reaction resulting from the de-eth-
3.3.2. Inactivation with DEP oxyformylatin of histidine residues (Srivastava & Kayastha, 2001).
DEP readily reacts with imidazole groups and hence are known Computation of a number of histidine groups modified by DEP at
to modify histidine residues with considerable specificity in pH different time intervals showed that at the initial stages the loss
range (6–7). This reaction proceeds with an increase in absorbance of activity was linearly related with moles of histidine residues
at 242 nm and the formation of an inactive ethoxyformylated blocked (1:1) (Fig. 5D) but later on, this ratio increases thus indi-
enzyme derivative. Wheat a-amylase modification with DEP cating that the active site histidine is the most reactive, is modified
resulting in enzymic inactivation was both time and modifier con- first, and as its availability decreases, other histidine residues get
centration dependent (data not shown). The linearity of plot of modified (Dua & Kochhar, 1985). On extrapolating the line, the %
log% residual activity against time using linear regression analysis loss in activity against the modified histidine residues, it can be
indicates that inactivation followed first-order kinetics with rate observed that only one histidine is involved at the active site.
constant (kobs) 0.112 min1. Fig. 5C shows that a-amylase lost its Referring to the accepted reaction mechanism of a-amylase
activity in a single exponential decay when incubated with catalysis ‘a-retaining double displacement’, glutamic acid is
0.5 mM DEP at pH 6.8 and 37 °C. Pre-incubation of enzyme with involved as acid/base catalyst and an aspartate as the nucleophile
starch for 5 min before DEP modification rendered significant attacking the glucose ring (Maarel & Veen, 2002). Clearly, the
8 K. Singh, A.M. Kayastha / Food Chemistry 162 (2014) 1–9

(A) 3.2 (B)

2.2
3.0 5 mg mL-1 starch + Enzyme 0.1 mM EDC
0.1 mM EDC + Enzyme
2.8 2.0

Log % Residual Activity

2.6
Log Vmax

1.8
2.4

1.6
2.2

2.0 1.4

1.8
1.2
1.6

4 5 6 7 8 1.0
0 5 10 15 20 25
pKa1 pH pKa2
Time (min)

(C) (D)
2.2 100
0.5 mM DEP + Enzyme
5 mg mL-1 starch + Enzyme + 0.5 mM DEP
2.0
80
Log % Residual Activity

% Relative activity
1.8

60
1.6

1.4
40

1.2

20
1.0
0 2 4 6 8 10 12 14 16 18
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5
Time (min) No. of histidine residues modified

Fig. 5. (A) Effect of pH on log Vmax for wheat a-amylase, (B) kinetics of inactivation of wheat a-amylase in absence and presence of starch (5 mg/mL) by EDC, (C) kinetics of
inactivation of wheat a-amylase in absence and presence of starch (5 mg/mL) by DEP and (D) number of histidine residues modified against the relative activity of wheat
a-amylase using increase in absorbance at 240 nm with inactivation of wheat a-amylase by DEP.

presence of histidine residue at the active site does not coincide 4. Conclusions
with its involvement in the mechanism but the loss of wheat a-
amylase activity upon DEP treatment is quite conducive with its The purified wheat a-amylase was found to be stable enzyme
role in substrate hydrolysis. It has been observed that in a-amy- over 2 months at 4 °C. Having a high kcat, this demonstrates its abil-
lase, the histidine residue is found conserved from microorganisms ity to hydrolyse starch efficiently. Its thermal stability further indi-
to mammals (Kumari et al., 2010), hence it is a reasonable specu- cates its potential for industrial applications, especially for SDS-
lation that during the course of evolution, histidine has acquired based detergent industries.
a unique role which is physiologically significant for the control
of starch digestion. Acknowledgements
The present conclusion is further strengthened by site-directed
mutagenesis studies which indicated that histidine modification, One of us (K.S.) would like to acknowledge the financial assis-
aligned within the catalytic site, drastically reduced the a-amy- tance from Council of Scientific and Industrial Research (CSIR),
lase hydrolytic activity (Søgaard, Kadziola, Haser, & Svensson, New Delhi in the form of Junior and Senior research fellowships
1993). Reports on its function as a transition state stabilizer dur- to carry out this work. We also wish to thank Prof. D. Dash, at
ing catalysis and that its replacement greatly diminishes the sta- the Department of Biochemistry, Institute of Medical Sciences,
bility of the transition state, suggest that its presence at the active Banaras Hindu University, for providing the necessary facilities
site being conserved and catalytic in nature (Bartlett, Porter, for the pI determination.
Borkakoti, & Thornton, 2002; Smith, Heschel, King, & Taubman,
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