Characterization of a maltose-forming α-amylase from an amylolytic lactic acid bacterium Lactobacillus plantarum S21

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

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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 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 b

a r t i c l e

i n f o

Article history: Received 9 January 2015 Received in revised form 18 June 2015 Accepted 18 June 2015 Available online 20 June 2015 Keywords: ␣-Amylase Maltose-forming ␣-amylase Broad pH stability Lactobacillus plantarum Amylolytic lactic acid bacteria

a b s t r a c t A maltose-forming ␣-amylase was purified from the culture supernatant of Lactobacillus plantarum S21 cultivated on starch. The enzyme is a monomer with a molecular mass of 95 kDa, its activity is Ca2+ independent, and the optimum of amylase activity was found at pH 5.0 and 45 ◦ C. A remarkable property 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 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 confirmed to be an amylase-encoding gene by sequence comparison. The amylase gene encodes a protein of 910 amino acids including a signal peptide sequence. Both the nucleotide and amino acids sequence shared more than 96% identity with the ␣-amylases from L. plantarum A6, L. manihotivorans LMG18010 and L. amylovorus NRRL B-4540, yet the properties of the enzyme showed some distinct differences to 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 retted cassava, cassava roots, maize sourdough and some traditional fermented foods [1,2]. These bacteria produce extracellular amylolytic enzymes for the degradation of starchy substrates serving their metabolism. Homofermentative ALAB produce mainly lactic acid directly from starch as their metabolic end product. Therefore, they are an alternative and attractive option for the production of lactic acid from starch without the addition of exogenous amylolytic 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 amylolytic 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 [email protected] (C. Khanongnuch). http://dx.doi.org/10.1016/j.molcatb.2015.06.010 1381-1177/© 2015 Published by Elsevier B.V.

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 playing 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 lactic 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 organism 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 concentrations 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 purification and its detailed characterization is of high relevance in order to understand this ability of high efficacy bioconversion of starch to

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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.

but with an elution rate of 0.1 mL/min. Active fractions were pooled, desalted and stored at −80 ◦ C for further characterization.

2. Materials and methods

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 Comfort, Eppendorf, Hamburg, Germany) for 10 min. The reaction was terminated and reducing sugars liberated from starch were determined by adding 100 ␮L of DNS reagent, mixed and incubated at 100 ◦ C on dry bath (AccublockTM , Labnet International, Inc., Edison, 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 liberating 1 ␮mol of reducing sugars (as glucose equivalents) per min under assay condition.

2.1. Microorganisms Lactobacillus plantarum S21 (KF836428) was isolated from Thai fermented rice noodle in northern Thailand and identified as previously 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 highest quality available from Sigma (Steinheim, Germany). Plasmids were extracted using the PureYieldTM Plasmid Miniprep System (Promega Corp., Madison, WI, USA). DNA fragments were purified 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 BioRad 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 composition 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 single 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 centrifugation 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% saturation; 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 dissolved in 20 mM Na-phosphate buffer pH 6.5, and dialyzed against the same buffer at 4 ◦ C until equilibrium. An equilibrated solution 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 purification system was operated by the ÄKTAprime plus unit (GE Healthcare Bio-Science). The amylase was eluted by a linear gradient 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, Billeria, 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

2.5. Assay of amylase activity

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. Denaturing 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 protein 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 Naphosphate 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 determined using the standard 10-min activity assay but varying the

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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 immediately 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 different 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, amylose, amylopectin and glycogen. Substrate concentrations were varied from 0.2 to 20 g/L for these four substrates, and assay conditions 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 performance 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

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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 B4540 (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 aforementioned 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 gradient 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, Vilnius, Lithuania). The resulting plasmid was transformed into E. coli NEB5␣ according to the instruction of the manufacturer. Transformants 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 Spectrometry (LC–ESI-MS/MS). The desired protein bands were cut out from an SDS-PAGE gel, digested and S-alkylated with iodoacetamide 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% solvent 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.

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Table 1 Purification of extracellular amylase from L. plantarum S21. Step

Total activity (Units)

Total protein (mg)

Specific activity (U/mg)

Purification (folds)

Recovery (%)

Crude enzyme Precipitation Q-sepharose FF Q-sepharose HP

5310 3330 1820 1210

298 215 10.2 2.6

17.8 15.5 178 468

1.0 0.9 10 27

100 63 34 23

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 extracellular 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 chromatography. 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 preparation 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 determined 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. manihotivorans 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).

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 envisaged applications, since it differs from the properties of amylases isolated from ALAB so far. For example, the amylase from L. manihotivorans 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 amylase 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

Fig. 1. SDS-PAGE (A), zymogram (A) and native-PAGE (C). M, protein molecular mass marker.

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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, amylopectin 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 values were obtained for amylose followed by starch, amylopectin and glycogen (Table 2). Moreover, no cross activity of pullulanase 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.

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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 (%)

Starch Amylose Amylopectin Glycogen

100 105.5 89 49.5

± ± ± ±

2.9 3 0.9 0.6

Km (mg/mL) 8.4 9.8 9.1 15.2

± ± ± ±

0.7 1 1 1.1

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 amylase 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 glucose 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 relative 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,

vmax (␮mol mL−1 min−1 ) 584 998 440 313

± ± ± ±

21 58 21 13

kcat (s−1 )

kcat /Km (mL mg−1 s−1 )

1240 1680 980 487

148 172 108 32

amylose and amylopectin were 4.02, 4.77 and 4.02 mg/mL, respectively. 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 simultaneously 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 multiple 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]. Moreover, 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 oligosaccharides 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.

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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 comparable 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 various oligosaccharides from G2 to G7 [7]. Amylopullulanase from L. amylophilus GV6 was capable of hydrolysing pullulan to various amounts of glucose, maltose and maltotriose, but the gene encoding the enzyme has not been yet reported [12]. This preference 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 concentrations 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 maltosespecific 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 transport. Maltose phosphorylase specifically cleaves maltose to glucose and glucose-1-phosphate, which energetically is more favorable

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. manihotivorans 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 amylaseencoding gene was found to have two regions joined by a BamHI site (1422–1426 bp). The first region located upstream of this restriction site contains the active site and the conserved regions of ␣-amylases while the second one possesses 4 copies of a consensus 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 extracellular 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

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microorganisms such as Bacillus spp. shared only 50% identity or less. Functional analysis of the protein sequence for family classification 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 Nterminus. This peptide sequence contains four catalytic conserved regions that are found in other ␣-amylases. These regions of L. plantarum 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 hydrolysis 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 bioconversion 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 lactic acid fermentation. Thus, liquefying and saccharifying enzymes are a key factor affects lactic acid production particularly simultaneous 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 production 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 lactobacillal amylases, in particular stability over a broad range of pH. The pattern of hydrolysis products from the purified enzyme indicates that L. plantarum S21 ␣-amylase acts as both liquefying and saccharifying enzymes, and might be used for starch liquefaction

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