J. Microbiol. Biotechnol.

(2010), 20(9), 13071313

doi: 10.4014/jmb.1002.02005
First published online 13 July 2010

A New Protein of -Amylase Activity from Lactococcus lactis

Wasko, Adam*, Magdalena Polak-Berecka, and Zdzisl aw Targonski
Department of Biotechnology, Human Nutrition and Food Commodities, University of Life Sciences in Lublin, Poland, Skromna 8,
20-704 Lublin, Poland
Received: February 2, 2010 / Revised: April 21, 2010 / Accepted: June 2, 2010

An extracellular -amylase from Lactococcus lactis IBB500

was purified and characterized. The optimum conditions
for the enzyme activity were a pH of 4.5, temperature
of 35oC, and enzyme molecular mass of 121 kDa. The
genome analysis and a plasmid curing experiment indicated
that amy+ genes were located in a plasmid of 30 kb. An
analysis of the phylogenetic relationships strongly supported
a hypothesis of horizontal gene transfer. A strong homology
was found for the peptides with the sequence of amylases from Ralstonia pikettii and Ralstonia solanacearum.
The protein with -amylase activity purified in this study
is the first one described for the Lactococcus lactis species,
and this paper is the first report on a Lactococcus lactis
strain belonging to the amylolytic lactic acid bacteria
(ALAB).
Keywords: -Amylase, ALAB, Lactococcus lactis, gene
transfer, evolutionary origin

In developing countries, lactic acid bacteria (LAB) are mainly

used to improve the natural fermentation of foods with low
levels of readily fermentable sugars. Amylolytic lactic acid
bacteria (ALAB) capable of degrading starch in such food
matrixes as cereal grains or cassava play a beneficial role
in the fermentation processes [13]. To date, relatively few
ALAB have been isolated from starchy fermented foods
[36]. Various Lactobacillus strains exhibit amylase activity:
Lb. cellobiosus [38], Lb. amylovorus [24], Lb. amylophilus
[25], and Lb. amylolyticus [4]. Lb. plantarum and Lb.
manihotivorans have been isolated from cassava-based
fermented products [22, 27]. Amylolytic strains of Lb.
plantarum and Lb. fermentum have been isolated from
cereal-based fermented foods [1, 29, 36]. Amylolytic enzymes
from lactic acid bacteria have several applications in the starch
industry and are commercially important in the beverage,
food, and textile industries [12, 40]. Starch-degrading and
*Corresponding author
Phone: +48-81-46-23-355; Fax: +48-81-46-23-400;
E-mail: awasko1@tlen.pl

processing enzymes and the genetic engineering of the

microbes that produce them are of importance to the food,
chemical, and pharmaceutical industries.
In the earliest studies of bacterial -amylases, the enzymes
were described as monomers or homodimers ranging in
molecular mass from about 10 to 13.9 kDa, whereas typical
microbial -amylases are usually 50 to 60 kDa [41].
Further studies, however, reported high molecular masses
of the amylases, from 150 to 250 kDa [6], which was
confirmed by other authors. Imam et al. [15], for example,
reported that Lb. amylovorus produced an -amylase of a
high molecular mass (150 kDa), and similar results were
obtained by Burgess-Cassler and Imam [5].
Molecular studies of the -amylase genes of Lb. plantarum,
Lb. amylovorus, and Lb. manihotivorans show that there is
a more than 98% nucleotide sequence identity among
them. The high homology suggests that the three enzymes
possess similar structure-function relationships. It has been
demonstrated that the N-terminal parts of these enzymes
contain an active site. The 3'-terminal part is the region of
repeated sequences that might be responsible for rawstarch binding [33]. -Amylase genes (amyA) in Lactobacillus
strains are located mainly in chromosomal DNA [10, 11,
14]. On the other hand, genes encoding the putative
amylolytic enzymes (amyL, amyY, apu, and glgP) have
been found in the genome of Lactococcus lactis subsp.
lactis IL 1403, even though it has been proven that this
strain is unable to produce amylase enzymes [31].
In this report, we characterize a new protein with
amylolytic activity, isolated from a Lactococcus strain, and
describe the location of amy+ genes coding this protein.
We also make an attempt at explaining the phylogenetic
similarity of proteins from the -amylase family.
MATERIALS AND METHODS
Bacterial Strain and Culture Conditions
Lc. lactis IBB500, an amylolytic strain isolated from plants (kindly
provided by Prof. Bardowski, Institute of Biochemistry and Biophysics,

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Wasko et al.

Polish Academy of Sciences, Warsaw, Poland) was propagated in

BHI medium (Manual of BBL Products and Laboratory Procedures)
at 28oC for 24 h. Stock cultures were suspended in 15% glycerol
and deep-frozen at -80oC.
Fresh bacterial culture (100 ml) was inoculated into 1.9 dm3 of the
medium with soluble potato starch as a sole carbon source (10 g/l).
The fermentation was performed in a 2.5 dm3 stirred (150 rpm)
fermentor (New Brunswick Scientific-BIOFLO). Culture was performed
in anaerobic conditions at 28oC for 24 h at a constant pH (6.0) that
was kept by adding NaOH (5%). Samples were withdrawn throughout
the fermentation process, and the supernatant was analyzed for
enzyme activity.
Enzyme Activity Assay
Extracellular amylase activity was assayed based on the 3,5dinitrosalicylic acid (DNS) method [16]. The reaction mixture (800 l)
consisted of 0.5% amylose (Sigma, Germany) in 100 mM sodium
acetate buffer (pH 4.5) and 200 l of the culture filtrate. The reaction
was stopped after 30 min of incubation at 35oC, and the reducing
sugars were determined. One unit of enzyme activity was defined as
the amount of enzyme that released 1 mole of reducing sugars per
minute under the assay conditions described above.
The apparent Michaelis constant of -amylase was determined at
10 different concentrations of starch at its temperature and pH optima.
Kinetic parameters were calculated by fitting the initial velocities
and substrate concentrations to the Michaelis-Menten equation using
the program WILMAN 4 (Minnesota State University, 1985).
Effects of pH and Temperature
The amylase activity was determined at various pH values (ranging
from 2.0 to 9.0) in 0.1 M acetate buffer. To determine thermostabilities
of the enzyme, the samples were preincubated in 0.1 M acetate buffer
at the optimum pH and temperatures ranging from 20 to 50C for
1 h. The samples were then chilled on ice for at least 30 min, and
residual activity was determined at pH 4.5 and the temperature of
35oC using the DNS method described above.
To study the effects of ions on the enzyme activity, samples in
0.1 M acetate buffer with Ca2+, Fe2+, Mg2+, Zn2+, Co2+, Al3+, and Hg+
at a final concentration of 1 mM, and Na+ at a final concentration of
50 mM, were incubated for 1 h at 35oC. The enzyme activity was
measured by the DNS method. The activity of the enzyme assayed
in 100 mM sodium acetate buffer alone, at the optimum pH, was
taken to be 100%.
Enzyme Purification
The fermentation broth was collected by centrifugation at 16,000 g for
15 min at 4oC, and proteins were precipitated with 60% ammonium
sulfate. The precipitate was resuspended in 10 ml of 0.1 M sodium
acetate buffer, pH 6.0, and dialyzed against sodium acetate buffer
(0.01 M, pH 6.0). The dialyzed samples were applied to a DEAESepharose Fast Flow ion-exchange column (Biologic LP, Bio-Rad,
Richmond, CA, U.S.A.). The column was washed with 0.01 M TrisHCl buffer, pH 6.0, and eluted with the same buffer with a linear
gradient 0.6 M NaCl at a flow rate of 1 ml/min. The initially purified
enzymes (5 ml of sample) were placed on a chromatographic column
of a Polybuffer Exchanger PBE 96 (ion-exchange) equilibrated with
25 mM imidazol-HCl buffer, pH 7.4. The fraction with -amylase
activity was eluted with 200 ml of washing buffer (Polybuffer96),
pH 3.4, at a flow rate of 0.5 ml/min.

The molecular mass of the enzyme was determined by gel filtration

column chromatography (Biologic LP Bio-Rad, Richmond, CA, U.S.A.)
using a Sephadex G-150 2.5100 cm column (Amersham Pharmacia,
Uppsala, Sweden) equilibrated with 0.01 M Tris-HCl buffer, pH 4.5.
The standard proteins used for the calibration of molecular mass were
HMW and LMW (Amersham Pharmacia). The flow rate was 1.7 ml/h.
HPLC Analysis of Product Formation
The 200 l samples of purified enzyme were incubated with 1% of
amylose, and soluble starch. The reaction conditions were the same
as in the enzyme assay experiment above. After incubation, samples
were withdrawn, boiled for 10 min, and clarified by centrifugation at
4,000 g for 10 min. The supernatant was analyzed by HPLC using
an Aminex HPX-42A saccharide analysis column (Bio-Rad). The
column was maintained at 85oC, and elution of the sugars was
performed with pure water. The following sugars were used as
references: glucose (DP1), maltose (DP2), maltotriose (DP3), maltotetraose
(DP4), and maltopentaose (DP5).
Electrophoretic Analysis
Polyacrylamide gel electrophoresis was performed in 10% (w/v)
gels by the method of Laemmli [19]. The enzyme activity in the
gels was detected after renaturation using the technique of Lacks
and Springhorn [18]. Protein profiles could be observed in the gels
after staining with Coomassie Brillant Blue R-250 or silver.
Structural Protein Analysis
The samples obtained after gel filtration (to remove buffer) were
concentrated under vacuum and freeze-dried, and the amino acid
sequence was determined using LC-MS/MS (ESI-TRAP-QTOF).
Protein was digested with semiTrypsin (sequencing Grade Modified
Trypsin, Promega V5111). The N-terminal short sequence homology
of the peptides obtained after LC-MS/MS was analyzed using
protein NR databases (all nonredundant GenBank CDS translations
+RefSeq Proteins+PDB+SwissProt+PIR+PRF) with the Blastp algorithm
(protein-protein BLAST) [2] with the following parameters: substitution
matrix-PAM 30, penalty for the break being 9, penalty for the
elongation of break being 1.
Phylogenetic Reconstruction
Neighbor-joining trees were obtained using Geneious Basic 3
(Biomatters). The statistical significance of the amylases in the species
phylogenetic tree was accessed using the following bootstrapping
approach; sequences of each protein were separately aligned with
the CLUSTAL X program, with its default parameters [39].
Plasmid DNA Extraction and Electrophoresis
Plasmid DNA was extracted by alkaline extraction as described by
Sambrook and Russell [35] and separated by standard agarose gel
electrophoresis. In the plasmid curing experiment, strains were
grown at different sublethal temperatures, which generated plasmid
loss [21]. The amylase activity was examined in the supernatant
after culture of Lc. lactis IBB 500 amy-.

RESULTS AND DISCUSSION

Table 1 shows the characteristics of the enzyme purified in
our study. The molecular mass of amylase from Lc. lactis

A NEW PROTEIN WITH -AMYLASE ACTIVITY FROM LACTOCOCCUS LACTIS

1309

Table 1. Properties of -amylase from Lactococcus lactis IBB

500.
Physicochemical properties
pH optimum
Temp. optimum
pH stability
Thermostabilitya
Molecular mass (SDS-PAGE)
Molecular mass (GF)
Isoelectric point
Km
Vmax
Effect of ionsb

Enzyme location
amyA gene location

Values
4.5
35oC
4.0-5.5
60 min/35oC
119.56 kDa
121 kDa
7.4
1,710,12 mg/ml
3,140,13 M/mlmg
Inhibitors Hg+ (19), Mn+ (30),
Fe2+ (89), Mg2+ (94), Zn2+ (83),
Co2+ (69), Al3+ (53)
Slight activator Ca2+ (102)
Extracellular
Plasmid

a
After incubation at 35oC for 60 min, the activity of the enzyme was equal
to the activity of untreated enzyme (100%).
b
The relative amylase activity (%) after addition of various metal ions.

IBB500 was over 100 kDa. This polypeptide was approximately

twice as large as typical bacterial -amylases [28, 41]. A
high molecular weight is a typical feature of amylases
isolated from ALAB [11, 32], which is also true of the
amylolytic protein purified in our study (Fig. 2). The apparent
molecular mass was 121 kDa, this value being similar to
the -amylases from L. amylovorus, L. plantarum, and L.
amylophilus, which range from 100 to 140 kDa [11, 15,
30]. The purified -amylase exhibited an isoelectric point
of 7.4. This value is higher than those reported for other
microorganisms, such as Bacillus subtilis [23] and Clostridium
acetobutylicum [30]. To our knowledge, there are no other
reports on the isoelectric point of lactococcal amylases.

Fig. 1. Agarose gel (0.7%) electrophoresis of plasmid DNA from

Lc. lactis IBB500 amyA (line a) and amy-.

The transformants amy- lost plasmids of 30 kb (lines b, c) and 2 kb (lines c,

d) after the curing experiment, when they were cultivated at 37oC, 35oC,
and 33oC for 24 h.

The elution pattern showed a unique protein peak that

corresponded to the amylase activity of the enzyme. The
enzyme, purified to homogeneity, migrated as a single
SDS-PAGE band, which was active on the zymogram
(Fig. 2). The purification steps are summarized in Table 2.
The purification procedure yielded an -amylase with a
specific activity of 2,551.42 U/mg protein, exhibiting a
purification factor of 14.36 and yield of 2.13%.
The amylase from Lc. lactis IBB500 showed an optimum
activity at a lower pH (4.5) and a lower temperature (35oC)

Fig. 2. SDS-PAGE of purified -amylase stained with silver (A) and separation and detection by PAGE of extracellular -amylase
from culture supernatants of L. lactis IBB500 (B).

The gel (B) was incubated in 20 mM sodium phosphate buffer pH 4.5 containing 1% soluble starch at 35oC for 1 h. The enzyme activity was detected by
staining the gel with 500 mM I2 solution.

1310

Wasko et al.

Table 2. Purification steps for -amylase secreted by Lactococcus lactis IBB500.

Total protein
content (mg)

Sample name
Crude enzyme solution
NH4(SO)4 precipitation 0-60%
DEAE-Sepharose Fast Flow
Sephadex G-150

Total enzyme
activity (U)

Specific enzyme activity

(U/mg protein)

Purification factor
(fold)

Enzyme
recovery (%)

8,350
7,450
301.8
178.6

177.6
252.5
1,077.9
2,551.4

1
1.4
4.3
14.4

100
89.2
3.6
2.1

47.0
29.5
0.28
0.07

than reported for amylases isolated from other ALAB.

Amylases described in other papers exhibited an optimum
activity at pH 5.0-6.4 and at a temperature range from
40oC to 60oC [6, 7, 20, 32]. The examined enzyme was
stable at 35oC for 60 min, after which period the activity of
the enzyme was lost. Petrov et al. [31] had revealed the
ability of lactococci to synthesize amylase enzymes. Those
authors found that an amylase from Lc. lactis subsp. lactis
B84 was a cell-bound enzyme with a maximum activity at
45oC and an optimum pH of 5.4. However, those authors
did not report any detailed information about the
physicochemical properties and purification of the enzyme.
The enzyme purified in this study was strongly inhibited
by bivalent ions. In the presence of Hg2+, the activity of
amylase was the lowest (19%). Similarly, Pompeyo et al.
[32] reported that L. amylophilus was totally or partially
inhibited by bivalent cations. The examined amylase was
not enhanced by Ca2+, which increased the enzymatic
activity only slightly to 102% (Table 1). The same effect
Table 3. Reaction products of the purified -amylase toward
polysaccharides.
Substrate
Soluble starch
Amylose

End products formed (%)

G1

G2

G3

G4

8.1
7.8

49.2
44.6

26.6
32.5

0.71

G5

was observed for amylases from other lactobacilli [32].

This finding confirms the hypothesis about structural
stabilization of the protein molecule by calcium ions,
which has been described for the -amylase family [17].
None of the metal ions was required for catalytic activity.
The -amylase showed a Michaelis-Menten type kinetics
when hydrolyzing soluble starch. The Km value for starch
of strain Lc. lactis IBB500 -amylase was 1.71 mg/ml,
which is within the range of many amylases from other
ALAB (0.35 to 4.7 mg/ml) [12, 33]. The Vmax value at 35oC
was 3.14 M/mlmg hydrolyzed starch (Table 1).
An examination of the end-products obtained from
degradation of soluble starch and amylase confirmed the
hypothesis that the amylase purified from Lc. lactis
IBB500 was a starch converting enzyme belonging to the
-1,4-D-glucan glucanohydrolases (E.C.3.2.1.1) family. The
products of hydrolysis are shown in Table 3. The products
of amylolytic attack were mixed oligosaccharides including
mainly maltose and maltotriose. This pattern of products
indicates that Lc. lactis IBB500 produces an endo-splitting
activity like that of -amylase, which hydrolyzes internal
bonds randomly and produces similar products of hydrolysis
[38].

Gn
16.1
14.39

G1, Glucose; G2, maltose; G3, maltotriose; G4, maltotetraose; G5,

maltopentaose; Gn, over maltopentaose.

Fig. 3. A mass spectrum of multiple sets of fragments of the

AWAALGIGGFRC peptide.

Fig. 4. Homology of the -amylase from L. lactis IBB500 with

the peptide sequences of Ralstonia pikettii and Ralstonia
solanacearum.

A NEW PROTEIN WITH -AMYLASE ACTIVITY FROM LACTOCOCCUS LACTIS

Fig. 5. Dendrogram showing the relationships among the amino acid sequences of -amylases from different bacterial species.

1311

The dendrogram was generated with the CLUSTAL X program [39]. Bacterial -amylases were divided into seven clusters. Bacteria with a homology
between the analyzed amino acid sequences are marked with * (ALAB) and # (Ralstonia sp.).

1312

Wasko et al.

All ALAB described to date have genes encoding

amylolytic enzymes, located in chromosomal DNA [10,
11]. From our study, it can be concluded that the genes
encoding extracellular -amylase in Lc. lactis IBB500 are
located in the plasmid of 30 kb (Fig. 1). The starchfermenting ability of the examined strain was lost after
plasmid curing. The cells that had lost the plasmid of
30 kb were not able to synthesize amylase, whereas loss of
other plasmids (20 kb and 2 kb) did not affect the amylolytic
activity of the examined strain. The plasmid curing
experiment indicated that part of the fermentation abilities
of Lc. lactis IBB500 could be due to plasmid-borne
characters, which can be unstable. A similar result was
obtained by Guyot et al. [13] for Lc. manihotivorans.
Those authors reported that, after plasmid curing, strain
OND 32 lost 91% of its ability to ferment -galactoside.
Some authors have proposed that the plasmid DNA
fragment represents a gene-cassette, which is vital for the
adaptation of lactococci to the plant environment [8].
Many genes important for the growth of LAB have been
found on mobile elements such as plasmids or transposons.
It is commonly known that lactic acid bacteria have a
natural ability to be transformed by exogenous DNA [34],
which may suggest that the plasmids in the examined Lc.
lactis IBB500 strain may have come from another bacterium.
Based on the described experimental results, we conclude
that the amylolytic property in Lc. lactis IBB500 was
acquired through lateral gene transfer.
Digestion with trypsin, and MS/MS determination of the
structure of the fraction obtained from FPLC, yielded 1,957
peptide sequences (Fig. 3). The N-terminal sequences were
compared with those from the protein data bank (NCBI)
using the protein-BLAST algorithm. The obtained results,
however, were not sufficient for full identification of the
analyzed enzyme with the proteins from the NCBI database.
A strong homology was found with the sequences of amylases from Ralstonia pickettii and Ralstonia solanacearum
(Fig. 4). The analysis of the phylogenetic relationships
strongly supported the hypothesis of a horizontal gene
transfer between the remotely related bacteria. As shown
in Fig. 5, the groups 2, 3, and 7 contained amylases of
ALAB species. Moreover, the last one also included
proteins isolated from Ralstonia pickettii and Ralstonia
solanacearum.
The hypothesis of gene transfer is supported not only by
protein homology, but also by the localization of the genes
for amylase on the plasmid. Successful gene transfer
between bacteria requires, among others, colonization of
the same environmental niche. In the present case, the
donor and the recipient bacteria came from plant material.
Ralstonia solanacearum is used as a model microorganism
for horizontal gene transfer studies. Other investigators
have found in the genome of this bacterium hot spot

regions in which recombination might be preferentially

initiated [9, 25].
The results of the present study imply that Lc. lactis
IBB500 is a new species, described for the first time in this
paper as ALAB. The differences between this strain and
other bacteria from this group concern the encoding of the
amy+ gene and the physicochemical characteristics of their
amylase enzymes, especially the low temperature (35oC)
and the low pH (4.5) required for optimum amylase activity
in the former. The results indicate that the hypothesis about
horizontal gene transfer between Ralstonia and Lactococcus
is feasible, as it explains the strong homology between the
amino acid sequences of the -amylases of these two
species.
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