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Purification and characterization of a thermostable phytate resistant α-amylase from Geobacillus sp. LH8
International Journal of Biological Macromolecules 46 (2010) 27–36
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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Purification and characterization of a thermostable phytate resistant ␣-amylase from Geobacillus sp. LH8 Nasrin Mollania, Khosro Khajeh ∗ , Saman Hosseinkhani, Bahareh Dabirmanesh Department of Biochemistry, Faculty of Biological Science, Tarbiat Modares University, P.O. Box 14115-175, Tehran, Iran
a r t i c l e
i n f o
Article history: Received 14 July 2009 Received in revised form 12 October 2009 Accepted 16 October 2009 Available online 27 October 2009 Keywords: ␣-Amylase Phytate resistance Thermostability Geobacillus sp. Homology modeling
a b s t r a c t A thermophilic and amylolytic bacterium (LH8) was isolated from the hot spring of Larijan in Iran at 65 ◦ C. Identification of strain LH8 by 16S rDNA sequence analysis showed that LH8 strain belongs to the Geobacillus sp. with 99% sequence similarity with the 16S rDNA of Geobacillus thermodenitrificans. A new ␣-amylase (GA) was extracted from this strain and purified by ion-exchange chromatography. SDS-PAGE showed a single band with an apparent molecular mass of 52 kDa. The optimum temperature and pH were 80 ◦ C and 5–7, respectively. In the presence of Mn2+ , Ca2+ , K+ , Cr3+ and Al3+ , the enzyme activity was stimulated while Mg2+ , Ba2+ , Ni2+ , Zn2+ , Fe3+ , Cu2+ and EDTA reduced the activity. The Km and Vmax values for starch were 3 mg ml−1 and 6.5 mol min−1 , respectively. The gene encoding ␣-amylase was isolated and the amino acid sequence was deduced. Comparison of GA and other ␣-amylase amino acid sequences suggested that GA has conserved regions that were previously identified in ␣-amylase family but GA exhibited some substitutions in the sequence. Its phytate resistant is an important property of this enzyme. 5 and 10 mM phytic acid did not inhibit this enzyme. Therefore, features of phytate resistant ␣-amylase from Geobacillus sp. LH8 are discussed. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Enzymes involved in starch bioconversions are of major industrial interest and considerable attention has been focused on obtaining new enzymes with improved properties or new applications. Thermostable starch-hydrolyzing enzymes such as amylases, pullulanases and glucoamylases play an important role in food, chemical, and pharmaceutical industries [1–3]. ␣-Amylase (1,4␣-d-glucan glucanohydrolase [EC 3.2.1.1]) hydrolyzes the internal ␣-(1,4) glycosidic links in amylose and amylopectin to produce a less viscous solution with lower molecular mass products limited by ␣-(1,6) glycosidic bonds which form the branch points in the native starch molecule. ␣-Amylases isolated from thermophilic bacteria are thermostable and active at high temperature. Bacillus species such as Geobacillus stearothermophilus, Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquifaciens, Bacillus cereus, Bacillus globisporus and Bacillus alvei are known for production of starch-hydrolyzing enzymes. More recently, Ezeji and Bahl reported that G. thermodenitrificans could produce a phytic acid resistant ␣-amylase [4]. Phytic acid which is the principal stor-
Abbreviations: GA, ␣-amylase from Geobacillus sp. LH8; BAA, ␣-amylase from Bacillus amyloliquefaciens; BLA, ␣-amylase from Bacillus licheniformis; BStA, ␣amylase from Bacillus stearothermophilus. ∗ Corresponding author. Tel.: +98 21 88009730; fax: +98 21 88009730. E-mail address: [email protected] (K. Khajeh). 0141-8130/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ijbiomac.2009.10.010
age form of phosphorous in many plant tissues especially bran and seeds, is an inhibitor of ␣-amylase. This inhibitory effect is one of the main drawbacks in industrial use of ␣-amylase. In the present work, we have purified, characterized, cloned and sequenced a phytate resistant ␣-amylase from Geobacillus sp. LH8. The relationship between ␣-amylase amino acid sequence and the phytate-resistance behavior is discussed. 2. Materials and methods 2.1. Chemicals 3,5-Dinitrosalicylic acid (DNS), soluble potato starch and Tris were purchased from Sigma (St. Louis, MO). Restriction enzymes, T4 DNA ligase and alkaline phosphatase were obtained from Fermentas (Germany). Q and monoQ-Sepharose were provided by Pharmacia (Uppsala, Sweden). All other reagent chemicals were obtained from Merck (Darmstadt, Germany). 2.2. Microorganism and culture conditions Geobacillus sp. LH8 which was previously isolated from the hot springs of Larijan-Iran was cultured in Horikoshi II medium (1% potato starch, 0.5% yeast extract, 0.5% peptone, 0.2% KH2 PO4 and 0.02% MgSO4 ·7H2 O) [5]. pH was strictly controlled at 6.8 by HCl. 100 ml trace elements solution containing CaCl·2H2 O (10−3 M),
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FeSO4 ·7H2 O (5 × 10−4 M), MnCl2 ·4H2 O (7 × 10−3 M), ZnSO4 ·7H2 O (8.7 × 10−5 M), H3 BO3 (4 × 10−3 M), CuSO4 ·5H2 O (5 × 10−5 M), Na2 MoO4 ·2H2 O (5.2 × 10−5 M), Co (NO3 )2 ·6H2 O (7.9 × 10−5 M) and 0.25 ml of concentrated H2 SO4 was prepared. In order to increase the production of ␣-amylase 300 l of the prepared solution was added to 1 L of culture medium. Cultures were incubated at 65 ◦ C in an orbital incubator. Growth medium was gently stirred at 185 rpm to maintain homogeneity. 2.3. DNA isolation and phylogenetic analysis through 16S rDNA sequence analysis To determine the phylogenic relationship, 16S rDNA sequencing studies were carried out on the bacterium [6]. Preparation of LH8 genomic DNA was performed according to Sambrook et al. [12]. The 16S rRNA gene was amplified using universal forward (5 -AGT TTG ATC CTG GCT CAG-3 ) and reverse (5 -GGC/T TAC CTT GTT ACG ACT T-3 ) primers [7,8]. These primers are complementary to phylogenetically conserved regions of the 16S rDNA in Bacillus sp. A DNA thermal cycler (Eppendorf) was programmed as follows: (1) an initial denaturing temperature of 94 ◦ C for 5 min, (2) a run of 30 cycles with each cycle consisting of 45 s at 94 ◦ C, 45 s at 48 ◦ C and 90 s at 72 ◦ C and (3) 5 min final extension at 72 ◦ C. The amplified products were purified with DNA extraction kit (Fermentas), and then DNA sequencing was performed on both strands by SEQ-LAB (Germany). The phylogenic relationship of the new isolate was determined through comparing the sequencing data with annotated sequences of related Bacillus and Geobacillus (GenBank database of the National Center for Biotechnology Information (Bethesda, MD), www.ncbi.nlm.nih.gov/GenBank/). A phylogenetic tree was constructed by the ClustalW software. The LH8 16S rDNA sequence has been deposited in GenBank under accession number of DQ192572. 2.4. Enzyme purification 2.4.1. Ammonium sulphate precipitation and dialysis All steps were carried out at 4 ◦ C. The crude culture was precipitated with ammonium sulphate (at 80% saturation) by slow continuous stirring at 4 ◦ C for 5 h. The saturated solution was centrifuged and the pellet was dissolved in minimum amount of 20 mM Tris–HCl (pH 8.5) containing 2 mM CaCl2 and dialyzed against 20 mM Tris buffer (pH 7.4). The dialyzed crude enzyme was filtered through a sterile filter (Schleicher and Schuell, Dessel, Germany). 2.4.2. Ion-exchange chromatography Filtered crude was applied onto Q-Sepharose column at a flow rate of 1 ml min−1 , which was previously equilibrated with 20 mM Tris buffer, pH 7.2. The enzyme was eluted with a linear gradient of sodium chloride (0–0.5 M) in the same buffer. Protein was monitored by measuring the absorbance at 280 nm and 250 l aliquots from each fraction was assayed for amylase activity. The active fractions were pooled and concentrated by ultrafiltration with a molecular weight cut-off of 30 kDa (Amicon, Bevery, MA, USA). Then, the concentrated ␣-amylase solution was applied to the monoQ-Sepharose column, connected to FPLC (Amersham Biosciences, Uppsala, Sweden) and eluted with Tris buffer at a flow rate 1 ml min−1 . Fractions containing ␣-amylase activity were pooled and dialyzed against 20 mM Tris buffer, pH 7.4. To determine molecular mass of the purified enzyme, the protein was subjected to SDS-PAGE and the gel stained with coomassie brilliant blue R250 [9,10]. 2.5. Activity determination and specific activity staining of gels ␣-Amylase activity was assessed at 65 ◦ C using potato starch (1%) as a substrate in 20 mM Tris–HCl, pH 7.4 containing 10 mM
CaCl2 . The activity was determined using 3,5-dinitrosalicylic acid (DNS) method according to Bernfeld which is based on the reducing sugar concentration [11]. One unit of ␣-amylase activity is defined as the amount of enzyme that produces reducing sugar equivalent to 1 mol maltose (as the standard) per minute under the assay conditions. Protein concentration was determined by the Lowry method [10]. SDS gel electrophoresis was performed using 10% acrylamide gel according to Laemmli [9]. To reveal activity after SDS-PAGE, the gel was rinsed with deionized water and shaken in Triton X-100 2.5% (v/v) for 45 min and it was then washed in 0.2 M sodium acetate buffer (pH 5.5) at 40 ◦ C for 45 min. The rinsed gel was transferred to fresh buffer containing 1% soluble starch and the incubation temperature was shifted to 65 ◦ C for duration of 30 min. Upon applying the Lugol’s solution at ambient temperature, protein bands with amylolytic activity became visible as white bands against a dark blue background. 2.6. Thermal stability Thermal stability of Geobacillus sp. LH8 ␣-amylase was measured by incubating the enzyme at 75, 85 and 90 ◦ C for different time periods (5, 10, 30 and 60 min) and subsequently cooled on ice for 30 min. The residual activity of GA was measured according to Bernfeld method [11]. Thermal stability was also determined in the presence of calcium chloride and EDTA. It should be noted that a stability control reaction was set up without using any of these additives. 2.7. Cloning of ˛-amylase gene Genomic DNA was prepared as described by DNA extraction procedures [12]. The encoding gene of ␣-amylase was selectively amplified using specific primers. The sequences of these primers are represented here: forward primer: 5 -GCG AGC TCG TGC TAA CGT TTC ACC-3 and reverse primer: 5 -GACTCGAGT CAA GGC CAT GCC ACC-3 . These primers were designed based on the ␣-amylase gene of G. stearothermophilus. The PCR reaction was programmed and carried out as follows (1) an initial temperature of 93 ◦ C for 5 min (2) a run of 30 cycles, each consisting of 45 s at 93 ◦ C, 45 s at 66 ◦ C and 90 s at 72 ◦ C and (3) 5 min at 72 ◦ C to allow for the extension of any incomplete products. Amplified products were separated on 1% agarose gel and ∼1.8 kb length fragment were purified from the gel using the QIAquick gel extraction kit (Qiagen). Two different Escherichia coli strains were used: DH5␣ and BL21. pET24d was used as the cloning vector. Preparation of competent cells of E. coli and other experimental procedures used to construct the designed plasmid were carried out through standard protocols [12]. Transformation of recombinant plasmids into E. coli cells was performed by CaCl2 method and electroporation [13–15]. Expression vector pET24d (+) was digested with restriction endonucleases SacI and XhoI. DNA fragments were ligated using the T4 DNA ligase. Competent cells of DH5␣ were transformed and grown overnight at 37 ◦ C in LB broth containing kanamycin. Plasmid DNA was extracted from the cells, transformed into BL21 (expression vector) and then plated on LB-kanamycin medium. Transformation was carried out using electroporation [16]. Recombinant plasmids containing the ␣-amylase gene were selected based on restriction analysis and sent for DNA sequencing, performed by MWG Biotech using an automatic DNA sequencer (LiCor) [17]. 2.8. Sequence analysis and homology modeling Analysis and translation of the nucleotide sequences were performed with the tools available at the ExPASy Molecular Biology Server (www.expasy.ch). Pair wise and multiple amino acid sequence alignments were carried out using the BLAST and
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Table 1 Purification procedure of ␣-amylase from Geobacillus LH8 strain. Step
Total activity (U)
Crude extraction (NH4 )2 SO4 Precipitation Q-Sepharose MonoQ-Sepharose
1440 1201 740 144
Total protein (mg) 24.5 16 2.8 0.2
CLUSTALW programs, respectively [18,19]. The structure of the Geobacillus sp. LH8 was modeled with the protein homology modeling SWISS-MODEL server using the crystal structure of G. stearothermophilus ␣-amylase (PDB code 1HVX) as template (swissmodel.expasy.org). Analysis and comparison of the structures were carried out using Swiss-PdbViewer ver3.7 [20,21].
Specific activity (U mg−1 )
Yield (%)
Purification (fold)
59 75 264 800
100 83 51 10
1 1.3 4.5 13.6
plot against [I0 ] will be also linear and the intercept on the [I0 ] axis would be −Ki [22,23]. 3. Results and discussion 3.1. Isolation and identification of the strain LH8 by 16S rDNA sequence analysis
2.9. Fluorescence measurements The intrinsic fluorescence intensity is an excellent parameter to monitor the polarity of aromatic side chain environment in a protein. Fluorescence emission spectra of B. licheniformis ␣-amylase (BLA) and pure GA enzyme at various concentrations of phytic acid were recorded on a PerkinElmer luminescence spectrometer LS 50B. 20 g ml−1 of ␣-amylase was prepared in different concentrations of phytic acid in 50 mM Tris buffer, pH 7.4 containing 10 mM CaCl2 . The mixture was allowed to equilibrate for 5 min. Emission spectra were recorded in the range of 300–400 nm using an excitation wavelength of 280 nm. 2.10. The effect of phytate on ˛-amylase One of the ways in which inhibition of enzyme-catalyzed reactions can be discussed is in terms of a general scheme shown below:
It is assumed that the enzyme-containing complexes are in equilibrium with each other, i.e., that the breakdown of ES in order to generate product does not significantly disturb the equilibrium [22,23]. In the present work, all data obtained for a particular system were analyzed using mix model of enzyme inhibition as described in the following equation: 1
v0
=
(1 + ([I0 ]/KI )) Km (1 + ([I0 ]/Ki )) 1 + Vmax Vmax [S0 ]
where v0 is the initial reaction rate, Vmax the maximum reaction rate, Km the binding constant for the starch, and Ki and KI are the inhibition constants for binding of inhibitor to enzyme and enzyme–substrate complex, respectively. In this model, a mix inhibitor displays finite but unequal affinity for both the free enzyme and the ES complex; hence the dissociation constants from each of these enzyme forms must be considered in the kinetic analysis of these inhibitors. Ki and KI were determined using secondary plots as describes by following equations: 1 + ([I0 ]/KI ) 1 = Vmax Vmax Slope for inhibited reaction = slope for uninhibited reaction × (1 + [I0 ]/Ki ). Hence a secondary plot of 1/Vmax against [I0 ] will be linear and the intercept on the [I0 ] axis gives −KI . A graph of slope of primary
Thermophilic bacteria were isolated from Larijan hot spring in north of Iran. One of these strains named LH8 was able to grow at 40–80 ◦ C (with an optimum temperature of 65 ◦ C) and showed the high amylolytic activity in the plate test. The isolated strain LH8 was a spore-forming, motile, rod-shaped, aerobic and Gram-positive bacterium. According to Bergey’s Manual of Systematic Bacteriology [24], this strain should belong to the genus Bacillus. Ribosomal RNA molecules are essential elements in protein synthesis which are conserved in all living organisms and their sequences are successfully used in order to identify and phylogenetically classify the biodiversity of the microbial world. Sequencing of PCR amplified 16S rDNA was performed. Length of the amplification product was 1500 bp (data not shown). 16S rDNA sequences from Bacillus and Geobacillus species were obtained from National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov/) and multiple sequence alignment was performed with ClustalW version 1.82. The phylogenetic tree was constructed using neighbor-joining method (data not shown). Multiple alignment and phylogenetic tree showed that LH8 strain belongs to the genus Geobacillus with 99% sequence similarity with the 16S rDNA of G. thermodenitrificans. To reach a more definitive classification, additional characteristics, especially DNA–DNA hybridization data, should be considered. 3.2. Production, purification and biochemical characterization of the ˛-amylase To improve the production of ␣-amylase, Geobacillus sp. LH8 was grown at 65 ◦ C on Horikoshi II medium containing trace elements [5]. After 36 h, the crude enzyme was brought to 80% saturation with ammonium sulphate. Geobacillus sp. LH8 ␣-amylase (GA) was purified by ionic-exchange chromatography using Q- and monoQSepharose columns (Fig. 1a and b). The purification procedure is summarized in Table 1. The specific activity of amylase from Geobacillus sp. LH8 was 75 U mg−1 after ammonium sulphate precipitation. In the first chromatography step involving Q-Sepharose anion exchange the ␣-amylase was separated from most extracellular proteins. The large peak showed the amylolytic specific activity of 264 U mg−1 . After ion-exchange chromatography on monoQ-Sepharose column the enzyme was purified 13.6-fold with a yield of 10%, and the specific activity was determined to be 800 U mg−1 . The enzyme was purified to homogeneity on SDSPAGE and migrated with a molecular mass of 52 kDa (Fig. 1c). Due to zymogram and PAGE analysis, it was assumed that the enzyme was monomer in its native form. The molecular mass of the new enzyme is slightly lower than those of the B. licheniformis and B. amyloliquefaciens ␣-amylases (∼56 kDa). Three ␣-amylases with a molecular mass of 58, 98 and 184 kDa were detected in B. stearothermophilus
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N. Mollania et al. / International Journal of Biological Macromolecules 46 (2010) 27–36 Table 2 The effect of metal ions and some reagents on enzyme activitya . Metal ions or reagents
Relative activity (5 mM)
Relative activity (10 mM)
Control (no addition) Na+ Mg2+ Zn2+ Mn2+ Fe3+ Ni2+ Ca2+ K+ Cr3+ Ba2+ Al3+ Cu 2+ Co2+ EDTA Phytic acidb
1.00 1.00 0.6 0.75 1.2 0.3 0.8 1.4 1.01 1.00 0.9 1.00 1.00 1.2 0.52 1.02
1.00 1.00 0.4 0.64 1.4 0.1 0.6 2.00 1.05 1.4 0.8 1.2 0.86 0.7 0.21 0.98
a Enzyme assays were performed in the presence of different additives, all at 5 and 10 mM. The relative activity of the enzyme was compared in 20 mM of Tris buffer with different chloride salts of Na+ , K+ , Mn2+ , Co2+ , Ba2+ , Ca2+ , Fe3+ , Al3+ , Zn2+ , Mg2+ , Cu2+ , Cr3+ , Ni2+ , EDTA and phytic acid. b 8, 12 and 15 mM of phytic acid were also examined and no inhibition was detected.
Fig. 1. (a) Anion-exchange chromatography on a Q-Sepharose column. The extracts (in 20 mM Tris–HCl buffer, pH 9.0) were applied to the column which was equilibrated with the same buffer. The enzyme was eluted with a NaCl gradient at a flow rate of 1 ml min−1 . (b) MonoQ-Sepharose chromatogram. Fractions with amylolytic activity were pooled and applied to the column which was previously equilibrated with 20 mM Tris buffer, pH 8.5. The ␣-amylase was eluted at 0–0.5 M NaCl at a flow rate of 1 ml min−1 . (c) SDS-PAGE analysis of samples from various purification steps. Proteins were detected by coomassie brilliant blue. The right panel is zymogram analysis. For more details please see Section 2.
DSM 2358 [25,26]. The mass of ␣-amylases from various microbial sources vary from 22.5 to 184 kDa [16,27]. The enzyme showed high activity in the pH range of 5–7 (Fig. 2a). The relative activity of GA at several temperatures (40–95 ◦ C) was determined using the standard procedure (Fig. 2b). The optimum temperature in the absence and presence of 10 mM CaCl2 was 70 and 80 ◦ C, respectively. The enzyme retained approximately 80% of its activity at 85 ◦ C. B. stearothermophilus ␣-amylase (BStA) opti-
mum temperature was reported in the range of 50–70 ◦ C. Optimum pH range of GA is comparable with most of Bacillus liquefying amylases, such as BAA, BStA, and BLA, that have an optimum pH between 5 and 7.5 [28]. GA has a good stability at high temperatures, which makes it remarkably heat stable for some industrial applications. The purified enzyme was dialyzed extensively against 20 mM Tris buffer (pH 7.4). Subsequently, the activity was measured in the presence of cations (5 and 10 mM) at 65 ◦ C. Mn2+ , Ca2+ , Cr3+ and Al3+ ions increased the enzyme activity, whereas Mg2+ , Ba2+ , Ni2+ , Zn2+ , Cu2+ , Fe3+ and EDTA decreased the activity. Furthermore, Na+ , K+ had no effect on ␣-amylase activity. ␣-Amylase activity was also investigated in the presence of different concentrations of phytic acid (5, 8, 10, 12, 15 mM) and no inhibitory effect was detected (Table 2). The time course of GA inactivation at 75, 80, 85 and 90 ◦ C was determined in 20 mM Tris–HCl, pH 7.4 in the absence and presence of 10 mM Ca2+ and 5 mM EDTA. As shown in Fig. 2c, 15% and 29% activity was seen after 10 min at 85 and 90 ◦ C, respectively. Thermal stability of the enzyme was influenced by the addition of Ca2+ and EDTA. Ca2+ strongly stabilized GA but EDTA, a chelating agent, reduced its stability (Fig. 2c). GA is completely inactivated after 6 min of incubation at 80 ◦ C in the presence of 5 mM EDTA. The requirement of calcium ion for thermal stability of other ␣amylases was also reported. For example, the liquefying enzymes from Bacillus strains require calcium ion for stability and enzyme activity [29,30]. ␣-Amylase inhibition by chelating compounds like EDTA shows that GA is a calcium metallo-enzyme; the same result has been reported for G. stearothermophilus ␣-amylases [31]. Kinetic parameters of GA on soluble starch were determined using Lineweaver-Burk plot. Km and Vmax values were 3 mg ml−1 and 6.5 mol min−1 , respectively (Fig. 2d). Although it is difficult to compare the kinetic values of amylases obtained by other workers due to different starch substrates and the assay conditions, the Km value of GA for starch (3 mg ml−1 ) was within range of many amylases (0.35–4.7 mg ml−1 ). 3.3. The nucleotide and deduced amino acid sequences of GA In order to obtain the nucleotide sequence of the gene encoding ␣-amylase, a set of primers were designed based on the sequence of ␣-amylase from G. stearothermophilus. After extraction of genomic DNA, the fragment was amplified with polymerase chain reac-
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Fig. 2. Influence of pH (a) and temperature (b) on activity of GA. The activity of purified enzyme was determined at various pHs and at different temperatures in the presence () and absence () of Ca2+ . (c) Thermostability of ␣-amylase. The purified enzyme was incubated at 75 ◦ C (), 80 ◦ C in presence of Ca2+ (䊉), 80 ◦ C without Ca2+ (×), 80 ◦ C in presence of EDTA (), 85 ◦ C () and 90 ◦ C () in Tris buffer, pH 7.5. After various periods of time, samples were withdrawn and the residual activity was measured with the standard assay. (d) Lineweaver-Burk plots and Michaelis-Menton curve (inset) for amylolytic activity of the enzyme. Standard deviations were within 6% of the experimental values. For more details please see Section 2.
tion. SacI and XhoI restriction sites were used in order to clone the amplified fragment into pET24d vector. Cloning was confirmed with PCR, double digestion and sequencing. Restriction analysis of recombinant clones revealed a 1.8 kb insert in pET24d vector. Subsequently, the nucleotide sequence of the inserted gene was analyzed and its amino acid sequence was determined. The gene (1650 bp) has an open reading frame of 549 amino acid residues. The deduced amino acid sequence was compared to known ␣amylases from Bacillus and Geobacillus species. The amino acid differences between GA, BStA, BLA and G6-amylase are shown in Fig. 3. It exhibits 83% and 56% identity to G. stearothermophilus ␣amylase (BStA) and B. licheniformis ␣-amylase (BLA) respectively. The model structure of GA was constructed with the crystal structure of G. stearothermophilus (PDB code: 1HVX) as template (Fig. 4) and the secondary structure predictions are shown in Table 3. The three-dimensional structure and topological fold of GA reveals similarity to other ␣-amylases family. It contains three domains, A, B and C, as first reported by Buisson et al. [32]. Domain A of this enzyme exhibit clear sequence similarities and a predicted common supersecondary fold, a parallel (␣/)8 -barrel [33–37]. The loop regions on the C-terminal side of the barrel generally are more complex than those of the N-terminal side and contain the active site and the calcium-binding site. Domain B, inserted between A3 and A␣3, form -sheets. Visual inspection of the sequences of domain B from representatives of the ␣-amylase family confirmed that domain B varies greatly in both length and sequence [37]. A distinct 103 amino acid long domain inserted between the strand 3 and helix ␣3 of the (␣/)8 -barrel in GA. BLA and BStA are different in length (105/102/101 residues for BLA/BStA/G6-Amylase). These enzymes have quite a long domain B in comparison with, e.g. the ␣-amylases from fungi (Aspergillus oryzae, 58 residues), animals (Homo sapiens pancreatic, 71 residues), plants (Hordeum vulgare, 61 residues), maltogenic ␣-amylases N-type (Thermus sp.
Table 3 Secondary structure elements of GA. Domain A A1 A␣1 A2 A␣2 A3 A␣3 A4 A␣4 A5 A␣5 A6 A␣6 A7 A␣7 A8 A␣8
Met42-Gln44 Ser56-Asn69 Ala74-Trp76 Lys115-Ala127 Gln131-Val136 Pro243-Thr260 Gly264-Leu267 Phe275-Thr289 Phe294-Gly297 ILe304-lys313 Ser319-Phe321 Ala323-Lys334 Ala357-Thr360 Lys381-Thr390 Arg394-Tyr399 Lys416-Asp426
Domain B B1 B2 B3 B4 B5 B6
His140-Lys141 Gly146-Ser154 Tyr168-lys175 Phe195-Asp201 Leu206-Phe212 Gly237-ILe239
Domain C C1 C2 C3 C4 C5 C6 C7 C8
Gly432-Tyr435 Ile442-Arg447 Leu457-Thr463 Gly468-Tyr473 Asn482-Thr485 Thr494-Asn495 Arg501-Tyr505 Val511-Val515
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Fig. 3. Multiple alignments of amino acid sequences of BLA, GA, BStA and G6-Amylase. In the case of BLA, the marked amino acid residues are involved in the CaI–Na–CaII metal triad binding. The CaIII binding occurs in the interface between domains A and C. The A under the sequences indicates the catalytic residues of ␣-amylases. The position of secondary structure elements, are indicated and extended areas which are suggested to contain a distinct thermoresponse (TSD) or substrate specificity determinant (G6G5) are marked by blue boxes. The sequence between residues 177 and 186 in BLA represents region I (♠) and residues from 255 to 270 indicate regions II (♣) as reported by Suzuki et al. [46] (BLA PDB code: 1BLI and BStA PDB code: 1HVX). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
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Fig. 4. Structural modeling of Geobacillus sp. ␣-amylase. The ribbon presentation of structural models were built using SWISS-MODEL server, based on crystal structure of Geobacillus stearothermophilus ␣-amylase (Protein Data Bank code: 1HVX).
IM6501, 55 residues) and saccharifying ␣-amylase (Bacillus subtilis, 49 residues). Domain C in all four proteins includes a so-called Greek key motif and forms a distinct globular unit [38]. Based on the comparison of eleven different ␣-amylases four stretches with the high degree of similarity were accepted as conserved regions [21]. The sequences comparison suggested that the amino acid sequence of GA includes four conserved regions (I through IV) that were previously identified in ␣-amylase family: (I) 135DVVFNH140, (II) 264GFRLDAVKH272, (III) 294FTVGEYW300, (IV) 361VDNHD365 (GA numbering). Region I contain three fairly conserved amino acids, Asp135, Asn139, and His140, which are important for the enzyme stability and activity. Region II contains the catalytic nucleophiles such as Asp268 and the invariant residue Arg266. These two residues were found in all ␣-amylases and are believed to be indispensable for catalytic activity. Regions III and IV contain Glu298 (catalytic proton donor) and Asp365 residue, respectively, which are conserved residues in these sites. Exhaustive analyses of a large number of amino acid sequences of ␣-amylase and related enzymes, proposed three additional conserved sequence regions. The fifth conserved sequence region, (V) 236NGDID240 (173 LPDLD in Taka-amylase A, 198YADID202 in BLA and 203YADID207 in G6-amylase), is located near the C-terminus of domain B around the conserved calcium-binding site, Asp238, in GA. This region appears to be the best conserved motif in domain B [32,33,39–42]. There are 30 residues between this Ca2+ -binding aspartate and the 4-strand catalytic aspartate, i.e., Asp268 in GA. This region in the form of QxDLN (x = P, A, I, W) is also found in ␣-glucosidase, dextran-glucosidase, trehalose-6-phosphate hydrolase, amylosucrase, sucrose phosphorylase, isomaltulose synthase and trehalose synthase [42]. Two additional conserved sequence regions (the regions VI and VII) can be found at strands 2 and 8 of the catalytic (␣/)8 -barrel domain [43,44]. Sixth conserved sequence region covering strand 2 of the catalytic (␣/)8 -barrel is 71GITALWLPP79 in GA. This 2-strand stretch, flanked in loops by a conserved glycine 71 and proline 79, could also be evolutionarily important for the (␣/)8 -barrel fold. This region in bacterial ␣-amylase (BLA), fungi ␣-amylase (Aspergillus oryzae), plant ␣-amylase (Hordeum vulgare), and G6amylase from Bacillus sp.707 is 36GITAVWIPP44, 56GFTAIWITP,
Fig. 5. Superimposition of the amino acid residues involved Ca I (a) and Ca III binding sites (b) in crystal structure of BLA, BStA and predicted structure of GA.
34GVTHVWLPP and 38GITAVWIPP46, respectively. The sixth conserved sequence region has been shown to be very helpful in discriminating, for example, the CGTases from ␣-amylases [42]. In order to distinguish CGTases from ␣-amylases, the length of this region has to be taken into account, together with special sequence features. The CGTases usually have eight residues between the Gly and Pro, while the ␣-amylases have seven [41]. GA has seven residues between the Gly71 and Pro79. The seventh conserved sequence region-strand 8 does not contain invariant residues, conserved throughout all the ␣-amylase family members, it usually starts with a very well-conserved glycine (Gly405 in GA in compared with Gly323 in Taka-amylase A, Gly368 in BLA and Gly373 in G6-amylase) followed by a proline in the i + 2 position (Pro407 in GA). It has been proposed that the conservations in region VI and VII may be related to maintenance of structure. In addition, according to the serial construction of ␣-amylase hybrids, Conrad et al. [45] have identified four regions from BLA which correlates with the thermostability and one region (G6G5) that affects the substrate specificity of ␣-amylases amylases. Furthermore, Suzuki et al. reported that regions I (177–186th position in BLA) and regions II (255–270th position in BLA) play a role in thermostability of the enzyme but the effect of region II is minor compared with that of region I [46]. Region I forms a loop on the surface of domain B in BLA. Similar loop with two extra residues exists in BStA and GA although there are differences between BStA and GA loop sequences (Fig. 3). Compared with other ␣-amylases from Bacillus and Geobacillus species, GA had some important substitutions in the sequence. The comparison exhibited a substitution (D139N) in the first calciumbinding site while G337D (between BStA, BLA and GA) and F339Y (BStA and GA) substitutions appeared in the third calcium-binding site (Fig. 5). Two other substitutions including Y136N and A147G
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Fig. 6. Superimposition of the amino acid residues in the active site in GA (red), BLA (green) and BStA (blue). AspA is 268 in GA, 231 in BLA and 234 in BstA, AspB is 365 in GA, 328 in BLA and 331 in BstA, and Glu is 298 in GA, 261 in BLA, and 264 in BstA (see Table 4). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
are also present in the newly identified fifth conserved region. Sequence alignment analysis illustrated in Fig. 3, indicates that Asp268, Glu298 and Asp365 (Fig. 6) are conserved catalytic residues at the corresponding positions [32,47]. They are positioned near the C-terminal of 4, 5, and 7 strands of the (␣/)8 -barrel domain. A number of residues in the vicinity of the conserved catalytically residues are believed to be conserved and play a role in the binding of substrate (Table 4). However, some differences in the 8 region of the catalytic (␣/)8 -barrel could be seen between BStA, BLA and newly isolated GA (Fig. 3). 3.4. The effect of phytic acid Phytic acid (known as inositol hexaphosphate (IP6) or phytate in its salt form), the principal storage form of phosphorus in many plant tissues especially in bran and seeds, is a selective inhibitor for ␣-amylases [48,49] and an anti-nutritional agent in monogastric animals [50]. Under normal physiological conditions, phytic acid chelates essential minerals such as calcium, magnesium, iron and zinc. It also interacts with amino acids and proteins over a wide pH range, forming phytate–protein complexes [51]. Results from this study suggest that phytic acid does not have an inhibitory effect on Geobacillus sp. LH8 ␣-amylase (GA). It was previously suggested that influence of ␣-amylase activity by phytic acid is due to its interactions with divalent cations (Ca2+ and Mg2+ ) [52]. Results of the present research explain that inactivation might be caused by other mechanisms, because GA activity
Fig. 7. Fluorescence intrinsic intensity spectra of ␣-amylase from B. licheniformis (BLA) at 20 mM Tris buffer, CaCl2 10 mM (pH 7.4) in the presence of various concentrations of phytic acid. The excitation wavelength was 280 nm with the enzyme concentration of 20 g ml−1 .
was suppressed with EDTA and the putative metal-binding sites were found in the constructed model. Probably as a consequence of the G337D substitution, an additional negative charge impeded the connection of phytic acid with the enzyme active site. To follow the structural changes of ␣-amylase from B. licheniformis (BLA), intrinsic fluorescence measurements were made in Tris 20 mM containing 10 mM CaCl2 in the presence of different concentrations of phytic acid. These measurements showed slight changes of the enzyme conformation in the presence of low concentrations of phytic acid. As the phytic acid concentration increased, tryptophan fluorescence gradually decreased (Fig. 7), suggesting a change in the tertiary structure of BLA and consequently the exposure of the buried tryptophan residues to the polar solvents. So, high concentrations of phytic acid readily disrupt the tertiary structures of BLA, which results in the reduction of its enzymatic activity (data
Table 4 Residues at and around the active site of GA. GA
BLA
BStA
Asp268 Asp365 Glu298 Gln44 Tyr91 Asp135 Val137 Lys271 Val296 Trp300 His364
Asp231 Asp328 Glu261 Gln9 Tyr56 Asp100 Val102 Leu234 Val259 Trp263 His327
Asp234 Asp331 Glu264 Gln10 Tyr57 Asp101 Val103 Lys237 Val262 Trp266 His330
Fig. 8. Lineweaver-Burk plots related to ␣-amylase inhibition at different concentrations of phytic acid. Concentration of the enzyme in all experiments is 20 g ml−1 . 1/v is the reciprocal initial velocity in unit−1 . Standard deviations were within 4% of the experimental values. For more details see Section 2.
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35
In summary, the thermostability of GA and its resistance to phytic acid (a selective ␣-amylase inhibitor), make this enzyme an interesting candidate for industrial applications. The results demonstrate that GA has a valuable selective advantage for corn processing and animal feed production since phytate is the major storage form of phosphate in cereals, legumes, pollen and oilseed. Therefore, it is of great value to employ genetic and fermentation techniques for the production of such enzymes on a large scale. Acknowledgments The authors express their gratitude to the Research Council of Tarbiat Modares University (Tehran Iran). We thank Dr. Janeˇcek for his critical comments regarding the manuscript. We also appreciate the laboratory assistance of Ms. Zarandi. References
Fig. 9. The secondary plots for the estimation of KI (a) and Ki (b). The data was obtained from Fig. 8. For more details see Section 2.
not shown). However, these findings and catalytic activity studies demonstrate a reduction in BLA activity without any major structural changes at low concentrations of phytic acid (0.01–0.05 mM). The same measurements were carried out using pure GA enzyme, and the results revealed no significant changes in the enzyme conformation (data not shown). Reaction rates were determined for different substrate concentrations using fresh enzyme samples each time and measurements were carried out at different starch concentrations in the presence of various concentrations of phytic acid (0.004, 0.01, 0.05 mM). The kinetic parameter values of BLA were determined from Lineweaver-Burk plots. Increasing the concentration of phytic acid caused a reduction in Km and Vmax values. The reciprocal plot related to the reversible inhibitory effect of phytic acid showed mixed inhibition patterns (Fig. 8). From secondary plots, Ki and KI values were 0.82 and 0.06 mM, respectively (Fig. 9a and b). Following the same pattern, phytate binds with greater affinity to the enzyme–starch complex or subsequent species in the reaction pathways. So, we would expect phytate exert a kinetic effect on the E + S → ES* process, thus effecting the apparent values of both Km and Vmax . We have to emphasize that GA was not inhibited at these concentrations of phytic acid.
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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Purification and characterization of a thermostable phytate resistant ␣-amylase from Geobacillus sp. LH8 Nasrin Mollania, Khosro Khajeh ∗ , Saman Hosseinkhani, Bahareh Dabirmanesh Department of Biochemistry, Faculty of Biological Science, Tarbiat Modares University, P.O. Box 14115-175, Tehran, Iran
a r t i c l e
i n f o
Article history: Received 14 July 2009 Received in revised form 12 October 2009 Accepted 16 October 2009 Available online 27 October 2009 Keywords: ␣-Amylase Phytate resistance Thermostability Geobacillus sp. Homology modeling
a b s t r a c t A thermophilic and amylolytic bacterium (LH8) was isolated from the hot spring of Larijan in Iran at 65 ◦ C. Identification of strain LH8 by 16S rDNA sequence analysis showed that LH8 strain belongs to the Geobacillus sp. with 99% sequence similarity with the 16S rDNA of Geobacillus thermodenitrificans. A new ␣-amylase (GA) was extracted from this strain and purified by ion-exchange chromatography. SDS-PAGE showed a single band with an apparent molecular mass of 52 kDa. The optimum temperature and pH were 80 ◦ C and 5–7, respectively. In the presence of Mn2+ , Ca2+ , K+ , Cr3+ and Al3+ , the enzyme activity was stimulated while Mg2+ , Ba2+ , Ni2+ , Zn2+ , Fe3+ , Cu2+ and EDTA reduced the activity. The Km and Vmax values for starch were 3 mg ml−1 and 6.5 mol min−1 , respectively. The gene encoding ␣-amylase was isolated and the amino acid sequence was deduced. Comparison of GA and other ␣-amylase amino acid sequences suggested that GA has conserved regions that were previously identified in ␣-amylase family but GA exhibited some substitutions in the sequence. Its phytate resistant is an important property of this enzyme. 5 and 10 mM phytic acid did not inhibit this enzyme. Therefore, features of phytate resistant ␣-amylase from Geobacillus sp. LH8 are discussed. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Enzymes involved in starch bioconversions are of major industrial interest and considerable attention has been focused on obtaining new enzymes with improved properties or new applications. Thermostable starch-hydrolyzing enzymes such as amylases, pullulanases and glucoamylases play an important role in food, chemical, and pharmaceutical industries [1–3]. ␣-Amylase (1,4␣-d-glucan glucanohydrolase [EC 3.2.1.1]) hydrolyzes the internal ␣-(1,4) glycosidic links in amylose and amylopectin to produce a less viscous solution with lower molecular mass products limited by ␣-(1,6) glycosidic bonds which form the branch points in the native starch molecule. ␣-Amylases isolated from thermophilic bacteria are thermostable and active at high temperature. Bacillus species such as Geobacillus stearothermophilus, Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquifaciens, Bacillus cereus, Bacillus globisporus and Bacillus alvei are known for production of starch-hydrolyzing enzymes. More recently, Ezeji and Bahl reported that G. thermodenitrificans could produce a phytic acid resistant ␣-amylase [4]. Phytic acid which is the principal stor-
Abbreviations: GA, ␣-amylase from Geobacillus sp. LH8; BAA, ␣-amylase from Bacillus amyloliquefaciens; BLA, ␣-amylase from Bacillus licheniformis; BStA, ␣amylase from Bacillus stearothermophilus. ∗ Corresponding author. Tel.: +98 21 88009730; fax: +98 21 88009730. E-mail address: [email protected] (K. Khajeh). 0141-8130/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ijbiomac.2009.10.010
age form of phosphorous in many plant tissues especially bran and seeds, is an inhibitor of ␣-amylase. This inhibitory effect is one of the main drawbacks in industrial use of ␣-amylase. In the present work, we have purified, characterized, cloned and sequenced a phytate resistant ␣-amylase from Geobacillus sp. LH8. The relationship between ␣-amylase amino acid sequence and the phytate-resistance behavior is discussed. 2. Materials and methods 2.1. Chemicals 3,5-Dinitrosalicylic acid (DNS), soluble potato starch and Tris were purchased from Sigma (St. Louis, MO). Restriction enzymes, T4 DNA ligase and alkaline phosphatase were obtained from Fermentas (Germany). Q and monoQ-Sepharose were provided by Pharmacia (Uppsala, Sweden). All other reagent chemicals were obtained from Merck (Darmstadt, Germany). 2.2. Microorganism and culture conditions Geobacillus sp. LH8 which was previously isolated from the hot springs of Larijan-Iran was cultured in Horikoshi II medium (1% potato starch, 0.5% yeast extract, 0.5% peptone, 0.2% KH2 PO4 and 0.02% MgSO4 ·7H2 O) [5]. pH was strictly controlled at 6.8 by HCl. 100 ml trace elements solution containing CaCl·2H2 O (10−3 M),
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FeSO4 ·7H2 O (5 × 10−4 M), MnCl2 ·4H2 O (7 × 10−3 M), ZnSO4 ·7H2 O (8.7 × 10−5 M), H3 BO3 (4 × 10−3 M), CuSO4 ·5H2 O (5 × 10−5 M), Na2 MoO4 ·2H2 O (5.2 × 10−5 M), Co (NO3 )2 ·6H2 O (7.9 × 10−5 M) and 0.25 ml of concentrated H2 SO4 was prepared. In order to increase the production of ␣-amylase 300 l of the prepared solution was added to 1 L of culture medium. Cultures were incubated at 65 ◦ C in an orbital incubator. Growth medium was gently stirred at 185 rpm to maintain homogeneity. 2.3. DNA isolation and phylogenetic analysis through 16S rDNA sequence analysis To determine the phylogenic relationship, 16S rDNA sequencing studies were carried out on the bacterium [6]. Preparation of LH8 genomic DNA was performed according to Sambrook et al. [12]. The 16S rRNA gene was amplified using universal forward (5 -AGT TTG ATC CTG GCT CAG-3 ) and reverse (5 -GGC/T TAC CTT GTT ACG ACT T-3 ) primers [7,8]. These primers are complementary to phylogenetically conserved regions of the 16S rDNA in Bacillus sp. A DNA thermal cycler (Eppendorf) was programmed as follows: (1) an initial denaturing temperature of 94 ◦ C for 5 min, (2) a run of 30 cycles with each cycle consisting of 45 s at 94 ◦ C, 45 s at 48 ◦ C and 90 s at 72 ◦ C and (3) 5 min final extension at 72 ◦ C. The amplified products were purified with DNA extraction kit (Fermentas), and then DNA sequencing was performed on both strands by SEQ-LAB (Germany). The phylogenic relationship of the new isolate was determined through comparing the sequencing data with annotated sequences of related Bacillus and Geobacillus (GenBank database of the National Center for Biotechnology Information (Bethesda, MD), www.ncbi.nlm.nih.gov/GenBank/). A phylogenetic tree was constructed by the ClustalW software. The LH8 16S rDNA sequence has been deposited in GenBank under accession number of DQ192572. 2.4. Enzyme purification 2.4.1. Ammonium sulphate precipitation and dialysis All steps were carried out at 4 ◦ C. The crude culture was precipitated with ammonium sulphate (at 80% saturation) by slow continuous stirring at 4 ◦ C for 5 h. The saturated solution was centrifuged and the pellet was dissolved in minimum amount of 20 mM Tris–HCl (pH 8.5) containing 2 mM CaCl2 and dialyzed against 20 mM Tris buffer (pH 7.4). The dialyzed crude enzyme was filtered through a sterile filter (Schleicher and Schuell, Dessel, Germany). 2.4.2. Ion-exchange chromatography Filtered crude was applied onto Q-Sepharose column at a flow rate of 1 ml min−1 , which was previously equilibrated with 20 mM Tris buffer, pH 7.2. The enzyme was eluted with a linear gradient of sodium chloride (0–0.5 M) in the same buffer. Protein was monitored by measuring the absorbance at 280 nm and 250 l aliquots from each fraction was assayed for amylase activity. The active fractions were pooled and concentrated by ultrafiltration with a molecular weight cut-off of 30 kDa (Amicon, Bevery, MA, USA). Then, the concentrated ␣-amylase solution was applied to the monoQ-Sepharose column, connected to FPLC (Amersham Biosciences, Uppsala, Sweden) and eluted with Tris buffer at a flow rate 1 ml min−1 . Fractions containing ␣-amylase activity were pooled and dialyzed against 20 mM Tris buffer, pH 7.4. To determine molecular mass of the purified enzyme, the protein was subjected to SDS-PAGE and the gel stained with coomassie brilliant blue R250 [9,10]. 2.5. Activity determination and specific activity staining of gels ␣-Amylase activity was assessed at 65 ◦ C using potato starch (1%) as a substrate in 20 mM Tris–HCl, pH 7.4 containing 10 mM
CaCl2 . The activity was determined using 3,5-dinitrosalicylic acid (DNS) method according to Bernfeld which is based on the reducing sugar concentration [11]. One unit of ␣-amylase activity is defined as the amount of enzyme that produces reducing sugar equivalent to 1 mol maltose (as the standard) per minute under the assay conditions. Protein concentration was determined by the Lowry method [10]. SDS gel electrophoresis was performed using 10% acrylamide gel according to Laemmli [9]. To reveal activity after SDS-PAGE, the gel was rinsed with deionized water and shaken in Triton X-100 2.5% (v/v) for 45 min and it was then washed in 0.2 M sodium acetate buffer (pH 5.5) at 40 ◦ C for 45 min. The rinsed gel was transferred to fresh buffer containing 1% soluble starch and the incubation temperature was shifted to 65 ◦ C for duration of 30 min. Upon applying the Lugol’s solution at ambient temperature, protein bands with amylolytic activity became visible as white bands against a dark blue background. 2.6. Thermal stability Thermal stability of Geobacillus sp. LH8 ␣-amylase was measured by incubating the enzyme at 75, 85 and 90 ◦ C for different time periods (5, 10, 30 and 60 min) and subsequently cooled on ice for 30 min. The residual activity of GA was measured according to Bernfeld method [11]. Thermal stability was also determined in the presence of calcium chloride and EDTA. It should be noted that a stability control reaction was set up without using any of these additives. 2.7. Cloning of ˛-amylase gene Genomic DNA was prepared as described by DNA extraction procedures [12]. The encoding gene of ␣-amylase was selectively amplified using specific primers. The sequences of these primers are represented here: forward primer: 5 -GCG AGC TCG TGC TAA CGT TTC ACC-3 and reverse primer: 5 -GACTCGAGT CAA GGC CAT GCC ACC-3 . These primers were designed based on the ␣-amylase gene of G. stearothermophilus. The PCR reaction was programmed and carried out as follows (1) an initial temperature of 93 ◦ C for 5 min (2) a run of 30 cycles, each consisting of 45 s at 93 ◦ C, 45 s at 66 ◦ C and 90 s at 72 ◦ C and (3) 5 min at 72 ◦ C to allow for the extension of any incomplete products. Amplified products were separated on 1% agarose gel and ∼1.8 kb length fragment were purified from the gel using the QIAquick gel extraction kit (Qiagen). Two different Escherichia coli strains were used: DH5␣ and BL21. pET24d was used as the cloning vector. Preparation of competent cells of E. coli and other experimental procedures used to construct the designed plasmid were carried out through standard protocols [12]. Transformation of recombinant plasmids into E. coli cells was performed by CaCl2 method and electroporation [13–15]. Expression vector pET24d (+) was digested with restriction endonucleases SacI and XhoI. DNA fragments were ligated using the T4 DNA ligase. Competent cells of DH5␣ were transformed and grown overnight at 37 ◦ C in LB broth containing kanamycin. Plasmid DNA was extracted from the cells, transformed into BL21 (expression vector) and then plated on LB-kanamycin medium. Transformation was carried out using electroporation [16]. Recombinant plasmids containing the ␣-amylase gene were selected based on restriction analysis and sent for DNA sequencing, performed by MWG Biotech using an automatic DNA sequencer (LiCor) [17]. 2.8. Sequence analysis and homology modeling Analysis and translation of the nucleotide sequences were performed with the tools available at the ExPASy Molecular Biology Server (www.expasy.ch). Pair wise and multiple amino acid sequence alignments were carried out using the BLAST and
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Table 1 Purification procedure of ␣-amylase from Geobacillus LH8 strain. Step
Total activity (U)
Crude extraction (NH4 )2 SO4 Precipitation Q-Sepharose MonoQ-Sepharose
1440 1201 740 144
Total protein (mg) 24.5 16 2.8 0.2
CLUSTALW programs, respectively [18,19]. The structure of the Geobacillus sp. LH8 was modeled with the protein homology modeling SWISS-MODEL server using the crystal structure of G. stearothermophilus ␣-amylase (PDB code 1HVX) as template (swissmodel.expasy.org). Analysis and comparison of the structures were carried out using Swiss-PdbViewer ver3.7 [20,21].
Specific activity (U mg−1 )
Yield (%)
Purification (fold)
59 75 264 800
100 83 51 10
1 1.3 4.5 13.6
plot against [I0 ] will be also linear and the intercept on the [I0 ] axis would be −Ki [22,23]. 3. Results and discussion 3.1. Isolation and identification of the strain LH8 by 16S rDNA sequence analysis
2.9. Fluorescence measurements The intrinsic fluorescence intensity is an excellent parameter to monitor the polarity of aromatic side chain environment in a protein. Fluorescence emission spectra of B. licheniformis ␣-amylase (BLA) and pure GA enzyme at various concentrations of phytic acid were recorded on a PerkinElmer luminescence spectrometer LS 50B. 20 g ml−1 of ␣-amylase was prepared in different concentrations of phytic acid in 50 mM Tris buffer, pH 7.4 containing 10 mM CaCl2 . The mixture was allowed to equilibrate for 5 min. Emission spectra were recorded in the range of 300–400 nm using an excitation wavelength of 280 nm. 2.10. The effect of phytate on ˛-amylase One of the ways in which inhibition of enzyme-catalyzed reactions can be discussed is in terms of a general scheme shown below:
It is assumed that the enzyme-containing complexes are in equilibrium with each other, i.e., that the breakdown of ES in order to generate product does not significantly disturb the equilibrium [22,23]. In the present work, all data obtained for a particular system were analyzed using mix model of enzyme inhibition as described in the following equation: 1
v0
=
(1 + ([I0 ]/KI )) Km (1 + ([I0 ]/Ki )) 1 + Vmax Vmax [S0 ]
where v0 is the initial reaction rate, Vmax the maximum reaction rate, Km the binding constant for the starch, and Ki and KI are the inhibition constants for binding of inhibitor to enzyme and enzyme–substrate complex, respectively. In this model, a mix inhibitor displays finite but unequal affinity for both the free enzyme and the ES complex; hence the dissociation constants from each of these enzyme forms must be considered in the kinetic analysis of these inhibitors. Ki and KI were determined using secondary plots as describes by following equations: 1 + ([I0 ]/KI ) 1 = Vmax Vmax Slope for inhibited reaction = slope for uninhibited reaction × (1 + [I0 ]/Ki ). Hence a secondary plot of 1/Vmax against [I0 ] will be linear and the intercept on the [I0 ] axis gives −KI . A graph of slope of primary
Thermophilic bacteria were isolated from Larijan hot spring in north of Iran. One of these strains named LH8 was able to grow at 40–80 ◦ C (with an optimum temperature of 65 ◦ C) and showed the high amylolytic activity in the plate test. The isolated strain LH8 was a spore-forming, motile, rod-shaped, aerobic and Gram-positive bacterium. According to Bergey’s Manual of Systematic Bacteriology [24], this strain should belong to the genus Bacillus. Ribosomal RNA molecules are essential elements in protein synthesis which are conserved in all living organisms and their sequences are successfully used in order to identify and phylogenetically classify the biodiversity of the microbial world. Sequencing of PCR amplified 16S rDNA was performed. Length of the amplification product was 1500 bp (data not shown). 16S rDNA sequences from Bacillus and Geobacillus species were obtained from National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov/) and multiple sequence alignment was performed with ClustalW version 1.82. The phylogenetic tree was constructed using neighbor-joining method (data not shown). Multiple alignment and phylogenetic tree showed that LH8 strain belongs to the genus Geobacillus with 99% sequence similarity with the 16S rDNA of G. thermodenitrificans. To reach a more definitive classification, additional characteristics, especially DNA–DNA hybridization data, should be considered. 3.2. Production, purification and biochemical characterization of the ˛-amylase To improve the production of ␣-amylase, Geobacillus sp. LH8 was grown at 65 ◦ C on Horikoshi II medium containing trace elements [5]. After 36 h, the crude enzyme was brought to 80% saturation with ammonium sulphate. Geobacillus sp. LH8 ␣-amylase (GA) was purified by ionic-exchange chromatography using Q- and monoQSepharose columns (Fig. 1a and b). The purification procedure is summarized in Table 1. The specific activity of amylase from Geobacillus sp. LH8 was 75 U mg−1 after ammonium sulphate precipitation. In the first chromatography step involving Q-Sepharose anion exchange the ␣-amylase was separated from most extracellular proteins. The large peak showed the amylolytic specific activity of 264 U mg−1 . After ion-exchange chromatography on monoQ-Sepharose column the enzyme was purified 13.6-fold with a yield of 10%, and the specific activity was determined to be 800 U mg−1 . The enzyme was purified to homogeneity on SDSPAGE and migrated with a molecular mass of 52 kDa (Fig. 1c). Due to zymogram and PAGE analysis, it was assumed that the enzyme was monomer in its native form. The molecular mass of the new enzyme is slightly lower than those of the B. licheniformis and B. amyloliquefaciens ␣-amylases (∼56 kDa). Three ␣-amylases with a molecular mass of 58, 98 and 184 kDa were detected in B. stearothermophilus
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N. Mollania et al. / International Journal of Biological Macromolecules 46 (2010) 27–36 Table 2 The effect of metal ions and some reagents on enzyme activitya . Metal ions or reagents
Relative activity (5 mM)
Relative activity (10 mM)
Control (no addition) Na+ Mg2+ Zn2+ Mn2+ Fe3+ Ni2+ Ca2+ K+ Cr3+ Ba2+ Al3+ Cu 2+ Co2+ EDTA Phytic acidb
1.00 1.00 0.6 0.75 1.2 0.3 0.8 1.4 1.01 1.00 0.9 1.00 1.00 1.2 0.52 1.02
1.00 1.00 0.4 0.64 1.4 0.1 0.6 2.00 1.05 1.4 0.8 1.2 0.86 0.7 0.21 0.98
a Enzyme assays were performed in the presence of different additives, all at 5 and 10 mM. The relative activity of the enzyme was compared in 20 mM of Tris buffer with different chloride salts of Na+ , K+ , Mn2+ , Co2+ , Ba2+ , Ca2+ , Fe3+ , Al3+ , Zn2+ , Mg2+ , Cu2+ , Cr3+ , Ni2+ , EDTA and phytic acid. b 8, 12 and 15 mM of phytic acid were also examined and no inhibition was detected.
Fig. 1. (a) Anion-exchange chromatography on a Q-Sepharose column. The extracts (in 20 mM Tris–HCl buffer, pH 9.0) were applied to the column which was equilibrated with the same buffer. The enzyme was eluted with a NaCl gradient at a flow rate of 1 ml min−1 . (b) MonoQ-Sepharose chromatogram. Fractions with amylolytic activity were pooled and applied to the column which was previously equilibrated with 20 mM Tris buffer, pH 8.5. The ␣-amylase was eluted at 0–0.5 M NaCl at a flow rate of 1 ml min−1 . (c) SDS-PAGE analysis of samples from various purification steps. Proteins were detected by coomassie brilliant blue. The right panel is zymogram analysis. For more details please see Section 2.
DSM 2358 [25,26]. The mass of ␣-amylases from various microbial sources vary from 22.5 to 184 kDa [16,27]. The enzyme showed high activity in the pH range of 5–7 (Fig. 2a). The relative activity of GA at several temperatures (40–95 ◦ C) was determined using the standard procedure (Fig. 2b). The optimum temperature in the absence and presence of 10 mM CaCl2 was 70 and 80 ◦ C, respectively. The enzyme retained approximately 80% of its activity at 85 ◦ C. B. stearothermophilus ␣-amylase (BStA) opti-
mum temperature was reported in the range of 50–70 ◦ C. Optimum pH range of GA is comparable with most of Bacillus liquefying amylases, such as BAA, BStA, and BLA, that have an optimum pH between 5 and 7.5 [28]. GA has a good stability at high temperatures, which makes it remarkably heat stable for some industrial applications. The purified enzyme was dialyzed extensively against 20 mM Tris buffer (pH 7.4). Subsequently, the activity was measured in the presence of cations (5 and 10 mM) at 65 ◦ C. Mn2+ , Ca2+ , Cr3+ and Al3+ ions increased the enzyme activity, whereas Mg2+ , Ba2+ , Ni2+ , Zn2+ , Cu2+ , Fe3+ and EDTA decreased the activity. Furthermore, Na+ , K+ had no effect on ␣-amylase activity. ␣-Amylase activity was also investigated in the presence of different concentrations of phytic acid (5, 8, 10, 12, 15 mM) and no inhibitory effect was detected (Table 2). The time course of GA inactivation at 75, 80, 85 and 90 ◦ C was determined in 20 mM Tris–HCl, pH 7.4 in the absence and presence of 10 mM Ca2+ and 5 mM EDTA. As shown in Fig. 2c, 15% and 29% activity was seen after 10 min at 85 and 90 ◦ C, respectively. Thermal stability of the enzyme was influenced by the addition of Ca2+ and EDTA. Ca2+ strongly stabilized GA but EDTA, a chelating agent, reduced its stability (Fig. 2c). GA is completely inactivated after 6 min of incubation at 80 ◦ C in the presence of 5 mM EDTA. The requirement of calcium ion for thermal stability of other ␣amylases was also reported. For example, the liquefying enzymes from Bacillus strains require calcium ion for stability and enzyme activity [29,30]. ␣-Amylase inhibition by chelating compounds like EDTA shows that GA is a calcium metallo-enzyme; the same result has been reported for G. stearothermophilus ␣-amylases [31]. Kinetic parameters of GA on soluble starch were determined using Lineweaver-Burk plot. Km and Vmax values were 3 mg ml−1 and 6.5 mol min−1 , respectively (Fig. 2d). Although it is difficult to compare the kinetic values of amylases obtained by other workers due to different starch substrates and the assay conditions, the Km value of GA for starch (3 mg ml−1 ) was within range of many amylases (0.35–4.7 mg ml−1 ). 3.3. The nucleotide and deduced amino acid sequences of GA In order to obtain the nucleotide sequence of the gene encoding ␣-amylase, a set of primers were designed based on the sequence of ␣-amylase from G. stearothermophilus. After extraction of genomic DNA, the fragment was amplified with polymerase chain reac-
N. Mollania et al. / International Journal of Biological Macromolecules 46 (2010) 27–36
31
Fig. 2. Influence of pH (a) and temperature (b) on activity of GA. The activity of purified enzyme was determined at various pHs and at different temperatures in the presence () and absence () of Ca2+ . (c) Thermostability of ␣-amylase. The purified enzyme was incubated at 75 ◦ C (), 80 ◦ C in presence of Ca2+ (䊉), 80 ◦ C without Ca2+ (×), 80 ◦ C in presence of EDTA (), 85 ◦ C () and 90 ◦ C () in Tris buffer, pH 7.5. After various periods of time, samples were withdrawn and the residual activity was measured with the standard assay. (d) Lineweaver-Burk plots and Michaelis-Menton curve (inset) for amylolytic activity of the enzyme. Standard deviations were within 6% of the experimental values. For more details please see Section 2.
tion. SacI and XhoI restriction sites were used in order to clone the amplified fragment into pET24d vector. Cloning was confirmed with PCR, double digestion and sequencing. Restriction analysis of recombinant clones revealed a 1.8 kb insert in pET24d vector. Subsequently, the nucleotide sequence of the inserted gene was analyzed and its amino acid sequence was determined. The gene (1650 bp) has an open reading frame of 549 amino acid residues. The deduced amino acid sequence was compared to known ␣amylases from Bacillus and Geobacillus species. The amino acid differences between GA, BStA, BLA and G6-amylase are shown in Fig. 3. It exhibits 83% and 56% identity to G. stearothermophilus ␣amylase (BStA) and B. licheniformis ␣-amylase (BLA) respectively. The model structure of GA was constructed with the crystal structure of G. stearothermophilus (PDB code: 1HVX) as template (Fig. 4) and the secondary structure predictions are shown in Table 3. The three-dimensional structure and topological fold of GA reveals similarity to other ␣-amylases family. It contains three domains, A, B and C, as first reported by Buisson et al. [32]. Domain A of this enzyme exhibit clear sequence similarities and a predicted common supersecondary fold, a parallel (␣/)8 -barrel [33–37]. The loop regions on the C-terminal side of the barrel generally are more complex than those of the N-terminal side and contain the active site and the calcium-binding site. Domain B, inserted between A3 and A␣3, form -sheets. Visual inspection of the sequences of domain B from representatives of the ␣-amylase family confirmed that domain B varies greatly in both length and sequence [37]. A distinct 103 amino acid long domain inserted between the strand 3 and helix ␣3 of the (␣/)8 -barrel in GA. BLA and BStA are different in length (105/102/101 residues for BLA/BStA/G6-Amylase). These enzymes have quite a long domain B in comparison with, e.g. the ␣-amylases from fungi (Aspergillus oryzae, 58 residues), animals (Homo sapiens pancreatic, 71 residues), plants (Hordeum vulgare, 61 residues), maltogenic ␣-amylases N-type (Thermus sp.
Table 3 Secondary structure elements of GA. Domain A A1 A␣1 A2 A␣2 A3 A␣3 A4 A␣4 A5 A␣5 A6 A␣6 A7 A␣7 A8 A␣8
Met42-Gln44 Ser56-Asn69 Ala74-Trp76 Lys115-Ala127 Gln131-Val136 Pro243-Thr260 Gly264-Leu267 Phe275-Thr289 Phe294-Gly297 ILe304-lys313 Ser319-Phe321 Ala323-Lys334 Ala357-Thr360 Lys381-Thr390 Arg394-Tyr399 Lys416-Asp426
Domain B B1 B2 B3 B4 B5 B6
His140-Lys141 Gly146-Ser154 Tyr168-lys175 Phe195-Asp201 Leu206-Phe212 Gly237-ILe239
Domain C C1 C2 C3 C4 C5 C6 C7 C8
Gly432-Tyr435 Ile442-Arg447 Leu457-Thr463 Gly468-Tyr473 Asn482-Thr485 Thr494-Asn495 Arg501-Tyr505 Val511-Val515
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N. Mollania et al. / International Journal of Biological Macromolecules 46 (2010) 27–36
Fig. 3. Multiple alignments of amino acid sequences of BLA, GA, BStA and G6-Amylase. In the case of BLA, the marked amino acid residues are involved in the CaI–Na–CaII metal triad binding. The CaIII binding occurs in the interface between domains A and C. The A under the sequences indicates the catalytic residues of ␣-amylases. The position of secondary structure elements, are indicated and extended areas which are suggested to contain a distinct thermoresponse (TSD) or substrate specificity determinant (G6G5) are marked by blue boxes. The sequence between residues 177 and 186 in BLA represents region I (♠) and residues from 255 to 270 indicate regions II (♣) as reported by Suzuki et al. [46] (BLA PDB code: 1BLI and BStA PDB code: 1HVX). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
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Fig. 4. Structural modeling of Geobacillus sp. ␣-amylase. The ribbon presentation of structural models were built using SWISS-MODEL server, based on crystal structure of Geobacillus stearothermophilus ␣-amylase (Protein Data Bank code: 1HVX).
IM6501, 55 residues) and saccharifying ␣-amylase (Bacillus subtilis, 49 residues). Domain C in all four proteins includes a so-called Greek key motif and forms a distinct globular unit [38]. Based on the comparison of eleven different ␣-amylases four stretches with the high degree of similarity were accepted as conserved regions [21]. The sequences comparison suggested that the amino acid sequence of GA includes four conserved regions (I through IV) that were previously identified in ␣-amylase family: (I) 135DVVFNH140, (II) 264GFRLDAVKH272, (III) 294FTVGEYW300, (IV) 361VDNHD365 (GA numbering). Region I contain three fairly conserved amino acids, Asp135, Asn139, and His140, which are important for the enzyme stability and activity. Region II contains the catalytic nucleophiles such as Asp268 and the invariant residue Arg266. These two residues were found in all ␣-amylases and are believed to be indispensable for catalytic activity. Regions III and IV contain Glu298 (catalytic proton donor) and Asp365 residue, respectively, which are conserved residues in these sites. Exhaustive analyses of a large number of amino acid sequences of ␣-amylase and related enzymes, proposed three additional conserved sequence regions. The fifth conserved sequence region, (V) 236NGDID240 (173 LPDLD in Taka-amylase A, 198YADID202 in BLA and 203YADID207 in G6-amylase), is located near the C-terminus of domain B around the conserved calcium-binding site, Asp238, in GA. This region appears to be the best conserved motif in domain B [32,33,39–42]. There are 30 residues between this Ca2+ -binding aspartate and the 4-strand catalytic aspartate, i.e., Asp268 in GA. This region in the form of QxDLN (x = P, A, I, W) is also found in ␣-glucosidase, dextran-glucosidase, trehalose-6-phosphate hydrolase, amylosucrase, sucrose phosphorylase, isomaltulose synthase and trehalose synthase [42]. Two additional conserved sequence regions (the regions VI and VII) can be found at strands 2 and 8 of the catalytic (␣/)8 -barrel domain [43,44]. Sixth conserved sequence region covering strand 2 of the catalytic (␣/)8 -barrel is 71GITALWLPP79 in GA. This 2-strand stretch, flanked in loops by a conserved glycine 71 and proline 79, could also be evolutionarily important for the (␣/)8 -barrel fold. This region in bacterial ␣-amylase (BLA), fungi ␣-amylase (Aspergillus oryzae), plant ␣-amylase (Hordeum vulgare), and G6amylase from Bacillus sp.707 is 36GITAVWIPP44, 56GFTAIWITP,
Fig. 5. Superimposition of the amino acid residues involved Ca I (a) and Ca III binding sites (b) in crystal structure of BLA, BStA and predicted structure of GA.
34GVTHVWLPP and 38GITAVWIPP46, respectively. The sixth conserved sequence region has been shown to be very helpful in discriminating, for example, the CGTases from ␣-amylases [42]. In order to distinguish CGTases from ␣-amylases, the length of this region has to be taken into account, together with special sequence features. The CGTases usually have eight residues between the Gly and Pro, while the ␣-amylases have seven [41]. GA has seven residues between the Gly71 and Pro79. The seventh conserved sequence region-strand 8 does not contain invariant residues, conserved throughout all the ␣-amylase family members, it usually starts with a very well-conserved glycine (Gly405 in GA in compared with Gly323 in Taka-amylase A, Gly368 in BLA and Gly373 in G6-amylase) followed by a proline in the i + 2 position (Pro407 in GA). It has been proposed that the conservations in region VI and VII may be related to maintenance of structure. In addition, according to the serial construction of ␣-amylase hybrids, Conrad et al. [45] have identified four regions from BLA which correlates with the thermostability and one region (G6G5) that affects the substrate specificity of ␣-amylases amylases. Furthermore, Suzuki et al. reported that regions I (177–186th position in BLA) and regions II (255–270th position in BLA) play a role in thermostability of the enzyme but the effect of region II is minor compared with that of region I [46]. Region I forms a loop on the surface of domain B in BLA. Similar loop with two extra residues exists in BStA and GA although there are differences between BStA and GA loop sequences (Fig. 3). Compared with other ␣-amylases from Bacillus and Geobacillus species, GA had some important substitutions in the sequence. The comparison exhibited a substitution (D139N) in the first calciumbinding site while G337D (between BStA, BLA and GA) and F339Y (BStA and GA) substitutions appeared in the third calcium-binding site (Fig. 5). Two other substitutions including Y136N and A147G
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N. Mollania et al. / International Journal of Biological Macromolecules 46 (2010) 27–36
Fig. 6. Superimposition of the amino acid residues in the active site in GA (red), BLA (green) and BStA (blue). AspA is 268 in GA, 231 in BLA and 234 in BstA, AspB is 365 in GA, 328 in BLA and 331 in BstA, and Glu is 298 in GA, 261 in BLA, and 264 in BstA (see Table 4). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
are also present in the newly identified fifth conserved region. Sequence alignment analysis illustrated in Fig. 3, indicates that Asp268, Glu298 and Asp365 (Fig. 6) are conserved catalytic residues at the corresponding positions [32,47]. They are positioned near the C-terminal of 4, 5, and 7 strands of the (␣/)8 -barrel domain. A number of residues in the vicinity of the conserved catalytically residues are believed to be conserved and play a role in the binding of substrate (Table 4). However, some differences in the 8 region of the catalytic (␣/)8 -barrel could be seen between BStA, BLA and newly isolated GA (Fig. 3). 3.4. The effect of phytic acid Phytic acid (known as inositol hexaphosphate (IP6) or phytate in its salt form), the principal storage form of phosphorus in many plant tissues especially in bran and seeds, is a selective inhibitor for ␣-amylases [48,49] and an anti-nutritional agent in monogastric animals [50]. Under normal physiological conditions, phytic acid chelates essential minerals such as calcium, magnesium, iron and zinc. It also interacts with amino acids and proteins over a wide pH range, forming phytate–protein complexes [51]. Results from this study suggest that phytic acid does not have an inhibitory effect on Geobacillus sp. LH8 ␣-amylase (GA). It was previously suggested that influence of ␣-amylase activity by phytic acid is due to its interactions with divalent cations (Ca2+ and Mg2+ ) [52]. Results of the present research explain that inactivation might be caused by other mechanisms, because GA activity
Fig. 7. Fluorescence intrinsic intensity spectra of ␣-amylase from B. licheniformis (BLA) at 20 mM Tris buffer, CaCl2 10 mM (pH 7.4) in the presence of various concentrations of phytic acid. The excitation wavelength was 280 nm with the enzyme concentration of 20 g ml−1 .
was suppressed with EDTA and the putative metal-binding sites were found in the constructed model. Probably as a consequence of the G337D substitution, an additional negative charge impeded the connection of phytic acid with the enzyme active site. To follow the structural changes of ␣-amylase from B. licheniformis (BLA), intrinsic fluorescence measurements were made in Tris 20 mM containing 10 mM CaCl2 in the presence of different concentrations of phytic acid. These measurements showed slight changes of the enzyme conformation in the presence of low concentrations of phytic acid. As the phytic acid concentration increased, tryptophan fluorescence gradually decreased (Fig. 7), suggesting a change in the tertiary structure of BLA and consequently the exposure of the buried tryptophan residues to the polar solvents. So, high concentrations of phytic acid readily disrupt the tertiary structures of BLA, which results in the reduction of its enzymatic activity (data
Table 4 Residues at and around the active site of GA. GA
BLA
BStA
Asp268 Asp365 Glu298 Gln44 Tyr91 Asp135 Val137 Lys271 Val296 Trp300 His364
Asp231 Asp328 Glu261 Gln9 Tyr56 Asp100 Val102 Leu234 Val259 Trp263 His327
Asp234 Asp331 Glu264 Gln10 Tyr57 Asp101 Val103 Lys237 Val262 Trp266 His330
Fig. 8. Lineweaver-Burk plots related to ␣-amylase inhibition at different concentrations of phytic acid. Concentration of the enzyme in all experiments is 20 g ml−1 . 1/v is the reciprocal initial velocity in unit−1 . Standard deviations were within 4% of the experimental values. For more details see Section 2.
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In summary, the thermostability of GA and its resistance to phytic acid (a selective ␣-amylase inhibitor), make this enzyme an interesting candidate for industrial applications. The results demonstrate that GA has a valuable selective advantage for corn processing and animal feed production since phytate is the major storage form of phosphate in cereals, legumes, pollen and oilseed. Therefore, it is of great value to employ genetic and fermentation techniques for the production of such enzymes on a large scale. Acknowledgments The authors express their gratitude to the Research Council of Tarbiat Modares University (Tehran Iran). We thank Dr. Janeˇcek for his critical comments regarding the manuscript. We also appreciate the laboratory assistance of Ms. Zarandi. References
Fig. 9. The secondary plots for the estimation of KI (a) and Ki (b). The data was obtained from Fig. 8. For more details see Section 2.
not shown). However, these findings and catalytic activity studies demonstrate a reduction in BLA activity without any major structural changes at low concentrations of phytic acid (0.01–0.05 mM). The same measurements were carried out using pure GA enzyme, and the results revealed no significant changes in the enzyme conformation (data not shown). Reaction rates were determined for different substrate concentrations using fresh enzyme samples each time and measurements were carried out at different starch concentrations in the presence of various concentrations of phytic acid (0.004, 0.01, 0.05 mM). The kinetic parameter values of BLA were determined from Lineweaver-Burk plots. Increasing the concentration of phytic acid caused a reduction in Km and Vmax values. The reciprocal plot related to the reversible inhibitory effect of phytic acid showed mixed inhibition patterns (Fig. 8). From secondary plots, Ki and KI values were 0.82 and 0.06 mM, respectively (Fig. 9a and b). Following the same pattern, phytate binds with greater affinity to the enzyme–starch complex or subsequent species in the reaction pathways. So, we would expect phytate exert a kinetic effect on the E + S → ES* process, thus effecting the apparent values of both Km and Vmax . We have to emphasize that GA was not inhibited at these concentrations of phytic acid.
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