Journal of Molecular Catalysis B: Enzymatic 120 (2015) 1–8

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Journal of Molecular Catalysis B: Enzymatic

journal homepage: www.elsevier.com/locate/molcatb

Characterization of a maltose-forming ␣-amylase from an amylolytic

lactic acid bacterium Lactobacillus plantarum S21
Apinun Kanpiengjai a , Saisamorn Lumyong b,c , Thu-Ha Nguyen d , Dietmar Haltrich d ,
Chartchai Khanongnuch a,c,∗
a
Division of Biotechnology, School of Agro-Industry, Faculty of Agro-Industry, Chiang Mai University, Chiang Mai 50100, Thailand
b
Microbiology Section, Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand
c
Material Science Center, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand
d
Food Biotechnology Laboratory, Department of Food Science and Technology, BOKU University of Natural Resources and Life Science, Vienna, Austria

a r t i c l e i n f o a b s t r a c t

Article history: A maltose-forming ␣-amylase was purified from the culture supernatant of Lactobacillus plantarum S21
Received 9 January 2015 cultivated on starch. The enzyme is a monomer with a molecular mass of 95 kDa, its activity is Ca2+ -
Received in revised form 18 June 2015 independent, and the optimum of amylase activity was found at pH 5.0 and 45 ◦ C. A remarkable property
Accepted 18 June 2015
of the enzyme is its stability over the broad pH range of 4.0–8.0 at 37 ◦ C, where 80–95% of its activity was
Available online 20 June 2015
retained for 12 days and 70–75% was retained for 30 days. The main hydrolysis products from starch,
amylose, amylopectin as well as glycogen were maltose (60%) and glucose (38%). The ORF of 2733 bp was
Keywords:
confirmed to be an amylase-encoding gene by sequence comparison. The amylase gene encodes a protein
␣-Amylase
Maltose-forming ␣-amylase
of 910 amino acids including a signal peptide sequence. Both the nucleotide and amino acids sequence
Broad pH stability shared more than 96% identity with the ␣-amylases from L. plantarum A6, L. manihotivorans LMG18010
Lactobacillus plantarum and L. amylovorus NRRL B-4540, yet the properties of the enzyme showed some distinct differences to
Amylolytic lactic acid bacteria these latter ␣-amylases and other lactobacillal ␣-amylases.
© 2015 Published by Elsevier B.V.

1. Introduction
Amylolytic lactic acid bacteria (ALAB) are lactic acid bacteria
capable of converting starchy biomass to lactic acid in a single
step. They have been isolated from various environments and
foods, particularly from amylaceous raw materials such as ret-
ted cassava, cassava roots, maize sourdough and some traditional
fermented foods [1,2]. These bacteria produce extracellular amy-
lolytic enzymes for the degradation of starchy substrates serving
their metabolism. Homofermentative ALAB produce mainly lactic
acid directly from starch as their metabolic end product. There-
fore, they are an alternative and attractive option for the production
of lactic acid from starch without the addition of exogenous amy-
lolytic enzymes. To select promising ALAB for the bioconversion of
starch to lactic acid, knowledge about the microorganisms and the
properties of their amylolytic enzymes are important. The amy-
lolytic enzyme from L. amylovorus NRRL B-4540, isolated from
cattle waste-corn fermentations, has been studied intensively in
∗ Corresponding author at: Division of Biotechnology, School of Agro-Industry,
Faculty of Agro-Industry, Chiang Mai University, Chiang Mai 50100, Thailand.
E-mail address: ck biot@yahoo.com (C. Khanongnuch).
the past [3–9]. Its properties are similar to those of the ␣-amylases
from L. plantarum A6 [10] and L. manihotivorans LMG18010 [2],
however efficient lactic acid production has not reported so far for
these strains. Most extracellular enzymes produced by ALAB play-
ing a role in bioconversion of starch to lactic acid have been found
to be ␣-amylases, amylopullulanases and pullulanases [11]. They
are expected to play an important role in the efficient production of
lactic acid from starch [12]. L. amylophilus GV6 is an amylolytic lac-
tic acid bacterium that effectively produced lactic acid from various
agricultural starchy residues [12–16]. At a level of 100 g/L soluble
starch, this strain was capable of producing up to 75.7 g/L of lactic
acid. The extracellular starch-degrading enzyme from this organ-
ism was classified as an amylopullulanase or maltrotriose-forming
␣-amylase [12]; however the enzyme-encoding gene has not yet
been reported. L. plantarum S21, an amylolytic lactic acid bacterium
recently isolated from Thai fermented rice noodles, was shown to
possess a good potential for the direct conversion of high concentra-
tions of starch, as it converted up to 100 g/L of starch to 94 g/L lactic
acid, which seems to be the highest production efficiency among
ALAB reported so far. The crude extracellular amylase has been
characterized in our previous study [17]; however, enzyme purifi-
cation and its detailed characterization is of high relevance in order
to understand this ability of high efficacy bioconversion of starch to

http://dx.doi.org/10.1016/j.molcatb.2015.06.010
1381-1177/© 2015 Published by Elsevier B.V.
2 A. Kanpiengjai et al. / Journal of Molecular Catalysis B: Enzymatic 120 (2015) 1–8
lactic acid. Therefore, this research describes the purification and
characterization of the extracellular amylase of L. plantarum S21.
The amino acid sequence of the amylase as well as the cloning and
sequencing of the amylase-encoding gene are also reported.
2. Materials and methods
2.1. Microorganisms
Lactobacillus plantarum S21 (KF836428) was isolated from Thai
fermented rice noodle in northern Thailand and identified as previ-
ously reported [17]. It was maintained in 15% glycerol and stored at
−80 ◦ C for further use. Escherichia coli NEB5␣ (New England Biolabs,
Ipswich, MA, USA) was grown in Lurian-Bertani (LB) medium.
2.2. Substrates and cloning kits
Soluble starch, amylose, amylopectin, ␣-cyclodextrin, ␤-
cyclodextrin, glycogen, dextrin, maltodextrin, dextran, pullulan
and maltooligosaccharides were analytical grade or of the high-
est quality available from Sigma (Steinheim, Germany). Plasmids
were extracted using the PureYieldTM Plasmid Miniprep System
(Promega Corp., Madison, WI, USA). DNA fragments were puri-
fied by the IllustraTM GFXTM PCR DNA and Gel Band Purification
Kit (GE Healthcare, Buckinghamshire, UK). Nucleic amplifications
were performed using Phusion High-Fidelity, GC buffer, dNTP mix,
oligonucleotide primers (VBC Biotech, Vienna, Austria) and a Bio-
Rad C-1000 thermocycler (BioRad, Vienna, Austria).
2.3. Media, culture condition, production of amylase and enzyme
preparation
Modified MRS medium (mMRS) was used for preparation of the
seed inoculum and as enzyme production medium. The composi-
tion was 10.0 g/L peptone, 10.0 g/L beef extract, 5.0 g/L yeast extract,
1.0 mL/L tween80, 2.0 g/L K2 HPO4 , 5.0 g/L CH3 COONa·3H2 O, 2.0 g/L
C6 H5 O7 (NH4 )2 H, 0.2 g/L MgSO4 ·7H2 O, 0.2/L g MnSO4 ·H2 O and
10.0 g/L soluble starch as the sole carbon source. The medium was
adjusted to pH 6.8 prior to sterilization (121 ◦ C for 15 min). A sin-
gle colony of L. plantarum S21 was inoculated into mMRS broth
and statically incubated at 37 ◦ C for 24 h. The mature culture was
transferred to the same medium and incubated under identical
conditions for 36 h. The culture supernatant was harvested by cen-
trifugation at 6371 × g at 4 ◦ C for 20 min and used as crude enzyme
for further purification.
2.4. Purification of amylase
Crude enzyme was precipitated by ammonium sulfate (80% sat-
uration; Carl Roth, Karlsruhe, Germany) at 4 ◦ C. The solution was
left at 4 ◦ C for 1 h before centrifugation at 6371 × g for 20 min.
The supernatant was discarded; the precipitated protein was dis-
solved in 20 mM Na-phosphate buffer pH 6.5, and dialyzed against
the same buffer at 4 ◦ C until equilibrium. An equilibrated solu-
tion was clarified by centrifugation in order to remove insoluble
particles prior to applying onto a 50 mL Q-sepharoseTM Fast Flow
(GE Healthcare Bio-Science, Uppsala, Sweden) column equilibrated
with binding buffer, 20 mM Na-phosphate buffer pH 6.5. The purifi-
cation system was operated by the ÄKTAprime plus unit (GE
Healthcare Bio-Science). The amylase was eluted by a linear gra-
dient of 0–0.5 M NaCl in 20 mM Na-phosphate buffer pH 6.5 with
flow rate of 10 mL/min. Active fractions were pooled, desalted using
10 kDa cut-off Amicon Ultra Centrifugal filter tubes (Millipore, Bil-
leria, MA, USA) prior to applying onto a 20 mL Q-sepharose High
performance (GE Healthcare Bio-Science) column for a polishing
step. Protein elution was performed as with Q-sepharose Fast Flow
but with an elution rate of 0.1 mL/min. Active fractions were pooled,
desalted and stored at −80 ◦ C for further characterization.
2.5. Assay of amylase activity
The reaction mixture consisted of 50 ␮L of appropriately diluted
enzyme in 100 mM Na-phosphate buffer pH 6.5 and 50 ␮L of 0.5%
(w/v) soluble starch in the same buffer. The reaction was carried
out at 37 ◦ C and 900 rpm on a rotary shaker (Thermomixer Com-
fort, Eppendorf, Hamburg, Germany) for 10 min. The reaction was
terminated and reducing sugars liberated from starch were deter-
mined by adding 100 ␮L of DNS reagent, mixed and incubated at
100 ◦ C on dry bath (AccublockTM , Labnet International, Inc., Edi-
son, NJ, USA) for 10 min. After cooling down, 800 ␮L of water was
added, mixed and the absorbance was measured at 540 nm. One
unit of amylase activity was defined as the amount of enzyme lib-
erating 1 ␮mol of reducing sugars (as glucose equivalents) per min
under assay condition.
2.6. Determination of protein concentrations
Protein concentrations were determined by the Bradford
method using a commercial kit (Protein assay system kit 600-0005,
BioRad) using bovine serum albumin as standard protein.
2.7. Polyacrylamide gel electrophoresis
SDS-PAGE was performed according to Laemmli [18] using a
10% resolving gel and a 4% stacking gel with 0.1% of SDS. Dena-
turing conditions were carried out by heating the protein solution
at 100 ◦ C for 4 min in the presence of ␤-mercaptoethanol using
2× Laemmli sample buffer (Sigma). Protein bands were detected
by staining with Bio-Safe Coomassie (BioRad, Hercules, CA, USA).
The precision Plus ProteinTM standard (BioRad) was used as pro-
tein molecular marker. For zymography analysis, a protein solution
containing 1 unit of amylase activity was heated at 70 ◦ C for 4 min
prior to loading onto the SDS-PAGE gel. After electrophoresis, the
gel was incubated with shaking in a cold solution of 20 mM Na-
phosphate buffer pH 6.5 for 30 min. Then it was immersed in 0.1%
(w/v) of soluble starch in 20 mM Na-phosphate buffer pH 6.5 for
5 min. The gel was rinsed with water and flooded with 0.3% (w/v)
I2 , 3% (w/v) KI in order to identify active protein bands, which were
visible as light bands on a dark blue background. Native PAGE was
performed using a 10% resolving gel and 4% stacking gel without
SDS, and using the HMW Calibration Kit (Amersham Bio-Sciences,
Uppsala, Sweden) as protein molecular mass marker. Protein bands
were stained and detected as described above.
2.8. Effect of pH and temperature on enzyme activity and stability
The pH optimum of amylase activity was determined using
standard assay conditions but varying the pH value from 3.0 to 9.0.
Citrate-phosphate buffer was used for pH 3.0–6.0, Na-phosphate
buffer was used for pH 6.0–8.0, and Tris–Cl was used for pH 8.0–9.0.
All buffer systems were prepared at a concentration of 100 mM.
In order to determine stability of amylase activity at different pH
values, a sample of purified amylase was incubated both at 4 and
37 ◦ C at various pH values ranging from 3.0 to 10.0 in 20 mM of
the appropriate buffers for 24 h. Residual activity was determined
under standard assay condition. In order to further monitor pH
stability at 37 ◦ C, the purified enzyme was incubated in 20 mM of
the appropriate buffers (pH 3.0–8.0) under aseptic condition for 30
days, and samples were taken frequently to measure the residual
activity.
The temperature optimum of amylase activity was deter-
mined using the standard 10-min activity assay but varying the
 A. Kanpiengjai et al. / Journal of Molecular Catalysis B: Enzymatic 120 (2015) 1–8
temperature from 25 to 65 ◦ C. To measure temperature stability,
the purified enzyme was incubated in 100 mM Na-phosphate buffer
pH 6.5 at 25, 30, 37, 45, 50, 55, 60 and 65 ◦ C for 1 h, and then imme-
diately placed on an ice bath. The residual amylase activity was
determined under standard assay conditions.
2.9. Effect of cations and chemicals on enzyme activity
Amylase activity was assayed in the presence of 5 mM K+ , Na+ ,
Li+ , Ag+ , Hg2+ , Ni2+ , Co2+ , Mg2+ , Cu2+ , Ba2+ , Mn2+ , Ca2+ , Pb2+ , Zn2+ ,
Ni2+ , Fe3+ , Al3+ , EDTA, SDS and 2-mercaptoethanol under otherwise
standard assay conditions. Relative activities are given compared
to that without any cations and chemical reagents added.
2.10. Substrate specificity
Amylase activity was assayed with 0.5% (w/v) of differ-
ent substrates including soluble starch, amylose, amylopectin,
␣-cyclodextrin, ␤-cyclodextrin, glycogen, dextrin, maltodextrin,
dextran and pullulan under standard assay conditions.
2.11. Determination of kinetic constants
The Km and vmax values of amylase from L. plantarum S21 were
determined for various substrates including soluble starch, amy-
lose, amylopectin and glycogen. Substrate concentrations were
varied from 0.2 to 20 g/L for these four substrates, and assay con-
ditions were otherwise identical to the standard assay (pH 6.5 and
37 ◦ C). The experimental data were fitted to the Michaelis–Menten
equation using SigmaPlot version 12.0 (Sysstat Software, Inc., San
Jose, CA, USA). The kcat value was defined as vmax /[E] where [E] is
enzyme concentration (␮mol/mL) used.
2.12. Determination of hydrolysis products
Purified amylase (1.0 U, corresponding to 0.2 U/mg substrate)
was incubated with 0.5% (w/v) of the following substrates: maltose
(G2), maltotriose (G3), maltotetraose (G4), maltopentaose (G5),
maltohexaose (G6), starch, amylose, amylopectin and glycogen
in Na-phosphate buffer pH 6.5 at 37 ◦ C for 24 h. The enzymatic
reaction was terminated by heating at 100 ◦ C for 10 min prior to
investigating the hydrolysis products by TLC. For HPLC analysis,
1.0 U of the purified amylase was incubated with 0.5% (w/v) of
starch, amylose and amylopectin in the same buffer. The reaction
was carried out at 37 ◦ C for 72 h and sample was taken periodically
for analysis.
2.13. Analysis of hydrolysis products by thin layer
chromatography (TLC)
One ␮L of each sample after hydrolysis was spotted on silica TLC
plates (Merck TLC silica gel 60, Damstadt, Germany) and dried by
heat. The plate was developed in a mobile phase system consisting
of n-butanol:ethanol:water in a ratio of 5:3:2 (v/v/v) at ambient
temperature for 4 h. For staining the plate was sprayed with 0.5%
(w/v) of thymol in 5% (v/v) of a sulfuric acid solution in ethanol and
heated at 105 ◦ C for 5 min to visualize spots in pink color.
2.14. Analysis of hydrolysis products by HPLC
The main hydrolysis products were quantified by high perfor-
mance anion exchange chromatography with pulsed amperometric
detection (HPAEC-PAD) using a Carbo Pac PA100 anion exchange
column (Dionex, Sunnyvale, CA, USA) equilibrated with 150 mM
NaOH. The separation was performed at 30 ◦ C using a linear
3
gradient of 500 mM Na-acetate, and sugars were detected by an
ED40 electrochemical detector using authentic G1–G6 as standards.
2.15. Isolation and sequencing of the amylase gene
A set of primers, including the forward primer AmyF 5 -
GTGAAAAAAAAGAAAAGTTTCTGG-3 and the reverse primer AmyR
5 -ATTAGGCTGGGCTGTTGTTG-3 , was designed according to a
sequence analysis of conserved regions of the amylase-encoding
genes of L. plantarum A6 (U62095), L. amylovorus NRRL B-
4540 (U62096) and L. manihotivorans LMG18010 (AF031369). The
genomic DNA of L. plantarum S21 was extracted using previously
described methods [17]. To isolate the amylase-encoding gene,
25 ␮L of standard PCR reaction was used together with the afore-
mentioned primers and the genomic DNA of L. planarum S21 as a
template. The PCR conditions were as follows: a pre-denaturation
step at 98 ◦ C for 2 min, 35 cycles of 10 s at 98 ◦ C, 20 s at a gra-
dient from 55 to 65 ◦ C and 60 s at 72 ◦ C, and a final cycle at
72 ◦ C for 7 min. The PCR product was applied onto an agarose
gel to observe the expected band. The blunt end fragment of the
amylase-encoding gene was purified from the gel, and inserted
into pJET/1.2 kb using the CloneJET PCR cloning kit (Fermentas, Vil-
nius, Lithuania). The resulting plasmid was transformed into E. coli
NEB5␣ according to the instruction of the manufacturer. Trans-
formants were plated on LB agar supplemented with 100 ␮g/mL
ampicillin. Positive clones were confirmed by restriction analysis
with XhoI digestion and commercial sequencing (LGC Genomics,
Berlin, Germany) using a set of primers consisting of the pJET1.2
forward and reverse sequencing primers and AmyF1 primer 5 -
AAGAACATCGATTTTCCATGGCAG-3 .
2.16. Determination of the amino acid sequence
The purified enzyme was analyzed by Liquid
Chromatography–Electrospray Ionization Tandem Mass Spec-
trometry (LC–ESI-MS/MS). The desired protein bands were cut
out from an SDS-PAGE gel, digested and S-alkylated with iodoac-
etamide in gel. Digestion was done with sequencing-grade trypsin
(Roche, Mannheim, Germany). About 3 ␮g of each digest was
loaded on a BioBasic C18 column (BioBasic-18, 150 × 0.18 mm,
5 ␮m, Thermo Fisher Scientific Inc., Waltham, MA, USA) using 0.1%
formic acid (FA) as the aqueous solvent. A gradient from 95% sol-
vent A (0.1% FA in water) and 5% solvent B (0.1% FA in acetonitrile)
to 32% B in 35 min was applied, followed by a 15-min gradient
from 32% B to 75% B that facilitates elution of large peptides at a
flow rate of 1.5 ␮L/min. Detection was performed with an Iontrap
MS (Bruker AmaZon ETD, Billerica, MA, USA) equipped with the
standard ESI source in the positive ion, DDA mode (= switching to
MSMS mode for eluting peaks). MS-scans were recorded (range:
500–1400 Da, target mass: 850) and the 8 highest peaks were
selected for fragmentation. Instrument calibration was performed
using ESI calibration mixture.
2.17. Sequence analysis
Sequence searches and similarity comparisons were performed
using the nucleotide blast and protein blast program from the
National Center for Biotechnology Information (NCBI) available at
http://blast.ncbi.nlm.nih.gov/Blast.cgi. Multiple alignments were
performed using the Genetyx-Win version 5.0.0 software (Genetyx
Co., Tokyo, Japan). Amino acid composition, theoretical isoelectric
point (pI) and calculated molecular mass were determined using
tools from the ExPASY Bioinformatics Resource Portal available
at http://web.expasy.org/compute pi/. The InterProScan tool was
used to analyze the amino acid sequence for family classification.
4 A. Kanpiengjai et al. / Journal of Molecular Catalysis B: Enzymatic 120 (2015) 1–8
Table 1
Purification of extracellular amylase from L. plantarum S21.
Step Total activity (Units) Total protein (mg)
Crude enzyme 5310 298
Precipitation 3330 215
Q-sepharose FF 1820 10.2
Q-sepharose HP 1210 2.6
3. Results and discussion
3.1. Enzyme production and purification
Production of amylase by L. plantarum S21 was performed by
cultivation in mMRS broth containing 10 g/L starch as the sole
carbohydrate source at 37 ◦ C for 48 h. The highest extracellu-
lar amylase activity of 2.1 U/mL (specific activity of 17.8 U/mg)
was obtained after 36 h of growth in this medium. Purification
of amylase was performed using a three-step protocol based on
precipitation by ammonium sulfate and anion-exchange chro-
matography. This yielded an amylase preparation that was purified
27-fold, with 23% recovery and a specific activity of 468 U/mg
(Table 1). The purification protocol resulted in an enzyme prepa-
ration of apparent homogeneity since both SDS-PAGE and native
PAGE gave only a single protein band. The purified L. plantarum
S21 amylase has an estimated molecular mass of 100 kDa as judged
from SDS-PAGE, and is a monomeric enzyme as concluded from the
active protein band on the zymogram as well as the mass deter-
mined by native PAGE (Fig. 1). This molecular mass is in the range
of that of other amylases of ALAB origin reported in the literatures,
which were shown to be monomeric enzymes with a molecular
mass of 135 kDa (L. plantarum A6) [10], 140 kDa (L. manihotivo-
rans LMG18010) [2], 150 kDa (L. amylovorus NRRL B-4540) [4], and
121 kDa (Lactococcus lactis IBB500) [19].
3.2. pH and temperature optimum and stability
The purified amylase retained more than 80% of its activity in the
pH range of 4.0–6.5 with the optimum activity at pH 5.0 (Fig. 2A).
Fig. 1. SDS-PAGE (A), zymogram (A) and native-PAGE (C). M, protein molecular mass marker.
Specific activity (U/mg) Purification (folds) Recovery (%)
17.8 1.0 100
15.5 0.9 63
178 10 34
468 27 23
More than 80% residual activity was observed when the enzyme
was incubated at 37 ◦ C in the pH range of 4.0–8.0 for 24 h (Fig. 2B),
and pronounced inactivation was only observed at pH 3.0 or 9.0
under these conditions. Furthermore, the L. plantarum amylase was
stable in the pH range of 4.0–8.0 when incubated at 37 ◦ C for 12
days (Fig. 2C), with more than 80% of the initial activity retained,
and 70–75% residual activity was obtained when incubated at these
conditions for 30 days. This broad pH stability in the range of 4.0–8.0
is an attractive feature of the L. plantarum S21 amylase for envis-
aged applications, since it differs from the properties of amylases
isolated from ALAB so far. For example, the amylase from L. mani-
hotivorans LMG18010 shows a pH optimum at 4.0–6.0 similar to
our enzyme, however with a much narrower pH range of stability
[2]. The optimum temperature for amylase activity was found at
45 ◦ C under the 10-min standard assay conditions (Fig. 3A). When
heated for 1 h at pH 6.5, the enzyme preparation retained close to
100% of its initial activity at temperatures ranging from 25 to 45 ◦ C
(Fig. 3B).
3.3. Effect of cations and chemicals
When added in concentrations of 5 mM, Mn2+ , Co2+ and Fe3+ ,
as well as 2-mercaptoehtanol showed an enhancing effect on amy-
lase activity by increasing the activity by 25–40% relative to when
no metal ion was added. Hg2+ in this concentration completely
inhibited the enzyme activity while Ag2+ inhibited the activity
significantly. All other cations and chemicals tested (K+ , Na+ , Li+ ,
Ni2+ , Mg2+ , Cu2+ , Ba2+ , Ca2+ , Al3+ , EDTA and SDS) showed either
no significant effect or a much lesser effect on amylase activity.
Notably, the addition of Ca2+ did not show any effect on amylase
 A. Kanpiengjai et al. / Journal of Molecular Catalysis B: Enzymatic 120 (2015) 1–8 5

Fig. 2. Effect of pH on amylase activity (A), stability at 37 ◦ C for 24 h (B), and stability at 37 ◦ C for 30 days (C). (A) Enzyme assay was conducted at different pH values (3.0–9.0)
and 37 ◦ C for 10 min, with the activity at pH 6.5 as 100%. (B) Enzyme activity was determined at pH 6.5 and 37 ◦ C for 10 min after incubation of the enzyme solution in
different pH values (3.0–9.0) at 37 ◦ C for 24 h. (C) Enzyme activity was determined at pH 6.5 and 37 ◦ C for 10 min after incubation at 37 ◦ C for 1, 2, 3, 5, 8, 12, 20 and 30 days.
The activity without incubation was set to 100%. The buffers used were citrate-phosphate (CP), sodium-phosphate (NP) and Tris–HCl (TC). The values are the mean of two
independent experiments.

activity, and hence this amylase is considered a Ca2+ -independent
enzyme.
3.4. Substrate specificity and steady-state kinetics
The amylase from L. plantarum S21 showed high activity
with amylose, soluble starch, amylopectin, maltodextrin and
glycogen, giving 105, 100, 89, 100, and 50% relative activity,
respectively (Table 2). The enzyme was unable to hydrolyze
raw starch, pullulan, ␣- and ␤-cyclodextrin under the chosen
reaction conditions. The Km values toward starch, amylose, amy-
lopectin and glycogen were in the range of 8.42–15.18 mg/mL at
37 ◦ C, pH 6.5, respectively. The highest vmax , kcat and kcat /Km val-
ues were obtained for amylose followed by starch, amylopectin
and glycogen (Table 2). Moreover, no cross activity of pullu-
lanase was found from the purified enzyme, hence the enzyme
is not of the amylopullulanase type that is frequently observed in
ALAB.

Fig. 3. Effect of temperature on amylase activity (A), and stability (B). (A) Enzyme assay was conducted at different temperature (25–65 ◦ C) and pH 6.5 for 10 min, with the
activity at 37 ◦ C as 100%. (B) Enzyme activity was determined at pH 6.5 and 37 ◦ C for 10 min after incubation at different temperatures (25–65 ◦ C) for 1 h. The activity without
incubation was set to 100%. The values are the mean of two independent experiments.
6 A. Kanpiengjai et al. / Journal of Molecular Catalysis B: Enzymatic 120 (2015) 1–8

Table 2
Substrate specificity and steady-state kinetics of purified amylase from L. plantarum S21 toward various substrates. The data given are the mean of two independent
experiments ± the standard deviation.

Substrates Rel. activity (%) Km (mg/mL) vmax (␮mol mL−1 min−1 ) kcat (s−1 ) kcat /Km (mL mg−1 s−1 )

Starch 100 ± 2.9 8.4 ± 0.7 584 ± 21 1240 148

Amylose 105.5 ± 3 9.8 ± 1 998 ± 58 1680 172
Amylopectin 89 ± 0.9 9.1 ± 1 440 ± 21 980 108
Glycogen 49.5 ± 0.6 15.2 ± 1.1 313 ± 13 487 32

3.5. Analysis of hydrolysis products
Defined oligosaccharides (G2–G6) as well as starch, amylose,
amylopectin and glycogen were used as substrates, and the main
products obtained after hydrolysis with the L. plantarum S21 amy-
lase were analyzed by TLC. Under the conditions chosen (0.2 U
of amylase activity per mg of substrate, 37 ◦ C, 24 h), maltose was
not cleaved by the enzyme, maltotriose was partly hydrolyzed to
maltose and glucose, while G4–G6 were completely hydrolyzed,
giving maltose as the main reaction product together with glu-
cose and maltotriose. Similar patterns were also obtained for the
polymeric substrates when analyzed by TLC (Fig. 4) with maltose
obtained as the main reaction product and only traces of the higher
oligosaccharides detected. Hydrolysis of the substrates including
soluble starch, amylose and amylopectin (each at 5 mg/mL) was
followed in more detail by HPLC (Fig. 5). Maltose was liberated
rapidly within 3 h from starch, amylose and amylopectin to 2.25,
2.58 and 2.13 mg/mL, corresponding to approximately 55% of the
total oligosaccharides formed, and then reached a constant value
of 2.43, 2.89 and 2.27 mg/mL, respectively. G3 was also formed
from these substrates in the initial phase of hydrolysis, but was
then degraded gradually until the end of hydrolysis, while the rel-
ative amount of G1 was increasing apparently to 1.50, 1.38 and
1.52 mg/mL (approximately 38% of the total oligosaccharides). G4
was detected in only low concentrations throughout the reactions.
The final concentration of reducing sugars obtained from starch,
amylose and amylopectin were 4.02, 4.77 and 4.02 mg/mL, respec-
tively.
Based on the results, we propose the mechanisms of ␣-amylase
from L. plantarum S21 as follows; the enzyme initially catalyzes
hydrolysis of polymeric substrates to produce large amount of G2
and G3 as the major and minor hydrolysis products, respectively.
It is suggested that the change of G3 to G1 could be simulta-
neously occurred by two mechanisms. Firstly, the G3 degradation,
G3 is hydrolyzed to G2 and G1 which mainly takes place in the
reactions similar to other ␣-amylases in the reported literature
[20]. Secondly, degradation of G2 to G1 in the presence of G3 as
a stimulator, this ␣-amylase might have a combination of mul-
tiple glycosyltransfer and/or condensation activity in addition to
hydrolysis activity among maltooligosaccharides to generate G1
according to the cyclic pathway of G2 degradation proposed by
Fujimori, et al. [21] and confirmed by Suganuma, et al. [22]. More-
over, it is suggested that there is an equilibrium concentration of G2
which may drive these two mechanisms. Whenever G2 is degraded
to G1, G3 will then be degraded to G2 and G1. Therefore, the G1 and
G3 concentration would be changed instead of G2 until running
out of G3. This explanation is in the agreement with oligosaccha-
rides obtained in Fig. 5. This study reveals that L. plantarum S21
␣-amylase is an endo-type enzyme but it acts as both liquefying
and saccharifying enzyme. However, the mechanism behind the
generation of high concentration of G1 and G2 will be our further
study.

Fig. 4. Hydrolysis products from various amylaceous substrates of purified amylase.

 A. Kanpiengjai et al. / Journal of Molecular Catalysis B: Enzymatic 120 (2015) 1–8
Fig. 5. HPLC analysis of maltooligosaccharides liberated from amylose (A), starch (B), and amylopectin (C), each at 5 mg/mL, during hydrolysis by purified amylase from L.
plantarum S21. Oligosaccharides are given as the percentage of the total oligosaccharides detected. The data presented are the mean of two independent experiments.
The pattern of hydrolysis products with maltose and glucose
as the main products is an attractive feature of the L. plantarum
S21 amylase. To date, very few maltose-forming amylases have
been reported among Lactobacillus sp. and other lactic acid bacteria.
Extracellular ␣-amylase from Streptococcus bovis 148 hydrolyzed
starch to 62.9% maltose and 37.1% glucose [23], which is com-
parable to the product pattern we observed for L. plantarum S21
amylase, while amylases from other Lactobacillus sp. such as L.
plantarum A6, L. manihotivorans, or L. amylovorus produced var-
ious oligosaccharides from G2 to G7 [7]. Amylopullulanase from
L. amylophilus GV6 was capable of hydrolysing pullulan to var-
ious amounts of glucose, maltose and maltotriose, but the gene
encoding the enzyme has not been yet reported [12]. This prefer-
ence of forming mainly maltose and glucose might also explain
our previous results, showing that L. plantarum S21 has a high
potential for the direct and rapid conversion of increased con-
centrations of starch to lactic acid [17,24]. The Gram-positive
bacterium Bacillus subtilis [25] takes up maltose by two different
systems, a phosphoenolpyruvate-dependent phosphotransferase
system (PTS) together with the maltose-specific enzyme IICB
and a maltodextrin-specific ABC transporter, composed of the
maltodextrin-binding protein MdxE, the membrane-spanning
components MdxF and MdxG, and the ATPase MsmX. The genome
of L. plantarum WCFS1 [26] does not indicate a putative maltose-
specific PTS system, yet the genes for the maltodextrin-specific ABC
transporter (mdxE, mdxF, mdxG, msmX) are present together with
two genes encoding maltose phosphorylases (mapA, mapB), which
lack a signal peptide suggesting a role in maltose metabolism in
the cytosol. In contrast to the PTS system, ABC systems do not
phosphorylate or otherwise modify their substrates during trans-
port. Maltose phosphorylase specifically cleaves maltose to glucose
and glucose-1-phosphate, which energetically is more favorable
7
than simple hydrolysis, while it shows only very low activity with
maltotriose or higher maltodextrins [27,28]. Hence an amylolytic
system that produces predominantly maltose could provide certain
energetic advantages to an organism.
3.6. Cloning of the amylase gene and confirmation of the amino
acid sequence
A complete open reading frame (ORF) for the amylase-encoding
gene consisting of 2733 bp (KJ440080) was isolated from L.
plantarum S21. This sequence shared highest identity with the
␣-amylase-encoding genes from L. plantarum A6 (U62095), L. mani-
hotivorans LMG18010 (AF031369) and L. amylovorus NRRL B4540
(U62096), and showed evidence for the conserved regions of
␣-amylases as described by Morlon-Guyot, et al. [29]. This amylase-
encoding gene was found to have two regions joined by a BamHI site
(1422–1426 bp). The first region located upstream of this restric-
tion site contains the active site and the conserved regions of
␣-amylases while the second one possesses 4 copies of a con-
sensus sequence [5,29]. The ORF of the amylase gene codes for
910 amino acid residues. The first 36 amino acids of this protein
were predicted by the SignalP-4.1 server (available at www.cbs.
dtu.dk/services/SignalP/) to be a signal peptide, and this was also
confirmed by amino acid sequencing of the purified, mature extra-
cellular enzyme by LC–ESI-MS/MS, which confirmed a polypeptide
of 874 amino acids starting with the sequence DSYT. A theoretical
molecular mass of 95.3 kDa and a pI of 4.41 were predicted by the
ExPASy server (available at http://web.expasy.org). The amino acid
sequence deduced from the complete gene shared 97, 96 and 91%
identity with the ␣-amylases from L. plantarum A6 (AAC45780), L.
manihotivorans LMG18010 (AAD45245), and L. amylovorus NRRL
B-4540 (AAC4578), respectively, while ␣-amylases from other
8 A. Kanpiengjai et al. / Journal of Molecular Catalysis B: Enzymatic 120 (2015) 1–8
microorganisms such as Bacillus spp. shared only 50% identity or
less. Functional analysis of the protein sequence for family clas-
sification by the InterProScan tool (available at www.ebi.ac.uk/
interpro) revealed that the L. plantarum S21 amylase belongs to
glycoside hydrolase family 13, with a structure based on a (␤/␣)8
barrel. The predicted catalytic residues of the enzyme were found
to conserve within the 441 amino acid residues located at the N-
terminus. This peptide sequence contains four catalytic conserved
regions that are found in other ␣-amylases. These regions of L. plan-
tarum S21 ␣-amylase show high similarity to that of the maltose
forming ␣-amylase from S. bovis 148 [23]. The identical regions are
region II (GFRYDAAKH) and region IV (WVESHD), whereas region
I and region III shows very high similarity. Hence, we classified it
to maltose-forming ␣-amylase. The N- and C-terminus are linked
by a flanking region that was also found in the L. plantarum A6
and L. manihotivorans LMG18010 amylases (TSSSSSSTTTET) called
the linker or 5 -end flanking region. Furthermore, four repeat units,
consisting of 91 amino acids each, are located at the C-terminal part
of the enzyme. These are connected by 3 intermediary regions (IRs),
which are rich in serine and threonine. These intermediary regions
are not completely conserved in the L. plantarum S21 amylase as is
the case of the IRs in the L. plantarum A6 amylase.
The primary sequence of the L. plantarum S21 ␣-amylase is
most similar to those of the three ␣-amylases from L. plantarum
A6, L. manihotivorans LMG18010 and L. amylovorus NRRL B-4540
[7] yet it shows some properties that are distinctly different from
those enzymes, namely the pH stability and the pattern of hydrol-
ysis products obtained after hydrolysis of polymeric substrates.
These distinct properties clearly explain the efficient direct lactic
acid production from starch by L. plantarum S21 in our previous
study [17,24]. Moreover, the enzyme is an attractive alternative to
related amylases from ALAB in term of applicability for bioconver-
sion of starch to lactic acid by lactic acid bacteria. In general, lactic
acid production from starch by lactic acid bacteria consists of at
least two steps; starch liquefaction and saccharification, and lac-
tic acid fermentation. Thus, liquefying and saccharifying enzymes
are a key factor affects lactic acid production particularly simulta-
neous liquefaction, saccharification lactic acid fermentation (SLSF).
This fermentation strategy is designated as a single step for lactic
acid production that both ␣-amylase and glucoamylase are added
together with lactic acid bacteria to discard the liquefaction and
saccharification step. However, these two enzymes normally work
under different pH condition [30] and are affected by the pH caused
from lactic acid produced during the fermentation. Therefore, the
pH adjustment is essentially required. Use of this broad pH stable
␣-amylase with maltose-forming activity is of high relevance to
avoid the pH adjustment steps and reduce glucoamylase used.
4. Conclusion
In this paper, we described the biochemical properties of a novel
amylase produced by L. plantarum S21. We selected this strain as
a source of an amylase because of its rapid and efficient produc-
tion of lactic acid from high concentrations of starch. L. plantarum
S21 amylase is typically found as the extracellular enzyme and
demonstrates some unique characteristics compared to other lac-
tobacillal amylases, in particular stability over a broad range of pH.
The pattern of hydrolysis products from the purified enzyme indi-
cates that L. plantarum S21 ␣-amylase acts as both liquefying and
saccharifying enzymes, and might be used for starch liquefaction
and saccharification step in order to produce fermentable sugars
for lactic acid production from starch.
Conflict of interest
The authors declare no conflict of interest.
Acknowledgements
The authors are grateful to for financial support from the ASEAN-
European Academic University Network (ASEA Uninet, http://
www.asea-uninet.org/) funded by the Austrian Federal Ministry
of Science, Research and Economy (BMWFW, http://www.bmwfw.
gv.at). This work was also supported by Postdoctoral fellowship
granted by Chiang Mai University.
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