Purification, Sequencing, and Biochemical ...

Purification, Sequencing, and Biochemical Characterization of a Novel CalciumIndependent α-Amylase AmyTVE from Thermoactinomyces vulgaris Ahmed K. A. El-Sayed, Mohamed I. Abou Dobara, Amira A. El-Fallal & Noha F. Omar Applied Biochemistry and Biotechnology Part A: Enzyme Engineering and Biotechnology ISSN 0273-2289 Appl Biochem Biotechnol DOI 10.1007/s12010-013-0201-7

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Author's personal copy Appl Biochem Biotechnol (2013) 170:483–497 DOI 10.1007/s12010-013-0201-7

Purification, Sequencing, and Biochemical Characterization of a Novel Calcium-Independent α-Amylase AmyTVE from Thermoactinomyces vulgaris Ahmed K. A. El-Sayed & Mohamed I. Abou Dobara & Amira A. El-Fallal & Noha F. Omar

Received: 18 February 2012 / Accepted: 18 March 2013 / Published online: 4 April 2013 # Springer Science+Business Media New York 2013

Abstract α-Amylase from Thermoactinomyces vulgaris was highly purified 48.9-fold by ammonium sulfate precipitation, gel filtration on Sephadex G-50 column, and ion exchange chromatography column of DEAE-cellulose. The molecular weight of the enzyme was estimated to be 135 and 145 kDa by SDS–PAGE. Its high molecular weight is due to high glycosylation. The purified amylase exhibited maximal activity at pH 6.0 to 7.0 and was stable in the range of pH 4.0 to 9.0. The optimum temperature for its activity was 50 °C. The enzyme half-life time was 120 min at 50 °C, suggesting intermediate temperature stable αamylase. The enzyme was sensitive to different metal ions, including NaCl, CoCl2, and CaCl2, and to different concentrations of EDTA. The enzyme activity was inhibited in the presence of 1 mM CaCl2, suggesting that it is a calcium-independent α-amylase. The TLC showed that the amylase hydrolyzed starch to produce large maltooligosaccharides as the main products. A 1.1-kb DNA fragment of the putative α-amylase gene (amy TVE) from T. vulgaris was amplified by using two specific newly designed primers. Sequencing analysis showed 56.2 % similarity to other Thermoactinomyces α-amylases with two conserved active sites confirming its function. Keywords α-Amylase . Purification . Characterization . DNA sequence . Thermoactinomyces vulgaris

Introduction The α-amylase (EC 3.2.1.1) is a well-known endoamylase that hydrolyzes starch by randomly cleaving internal α-1, 4-glucosidic linkages. The α-amylase has widely used in many fields, such as starch saccharification, textile, food, brewing, distilling industries, and medical and analytical chemistries [1]. α-Amylases have been isolated from diversified sources including plants, animals, and microbes, where they play a dominant role in A. K. A. El-Sayed : M. I. Abou Dobara : A. A. El-Fallal : N. F. Omar (*) Botany Department, Faculty of Science, Damietta University, P.O. Box 34517, New Damietta, Egypt e-mail: [email protected]

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carbohydrate metabolism. In spite of the wide distribution of α-amylase, microbial sources are used for the industrial production. Several different amylase preparations are available with various enzyme manufacturers for specific use in varied industries. However, the interest in new and improved α-amylase is growing vastly. The conditions prevailing in the industrial applications in which enzymes are used are rather extreme, especially with respect to temperature and pH. Therefore, there is a continuing demand to improve the stability of the enzymes and thus meet the requirements set by specific applications. In general, the search for thermostable Ca2+-independent αamylase [2] is required in most industries. However, a recent trend is to use intermediate temperature stable (ITS) α-amylases in backing industry [3]. Although a wide variety of microbial α-amylases is known, α-amylase with “ITS” property has been reported from only a few microorganisms [4]. Enzymes of the thermophilic actinomycetes Thermoactinomyces species, especially their α-amylase [5], have attracted much interest because of their activity at high temperature. In our previous study, we have determined the optimum conditions for production of highly active α-amylase from Thermoactinomyces vulgaris [6]. This enzyme showed calcium independency and satisfy stability in its crude form. The commercial use of α-amylase generally does not require purification of the enzyme, but enzyme applications in food industries, and pharmaceutical and clinical sectors require high purified amylases [7]. Also, the enzyme in purified form is a prerequisite in studies of structure–function relationships. Therefore, this study aims to purify and characterize the α-amylase from T. vulgaris on both physicochemical and molecular levels.

Materials and Methods T. vulgaris Strain The strain was isolated from a fertile soil samples collected from Egypt at 50 °C and identified according to Bergey’s Manual of Systematic Bacteriology [8]. The culture was maintained on Czapek-yeast-casein (CYC) agar slants. Production of α-Amylase The organism was grown in conical flasks containing starch– nitrate medium [9] as a basal medium containing equimolecular nitrogen amount of tryptone equivalent to the nitrogen content of the basal medium with pH adjusted to 7 and incubated at 55 °C with shaking (150 rpm) for 24 h. The cell-free enzyme supernatant was obtained by centrifugation at 8,000×g for 20 min. α-Amylase Assay α-Amylase assay was based on the reduction in blue color intensity resulting from starch hydrolysis [10]. One unit of enzyme activity is defined as the quantity of enzyme that caused 20 % reduction of blue color intensity of starch iodine solution at reaction incubation temperature in 1 min per milliliter. Protein Determination The Bradford method [11] was used to determine the protein concentration. Bovine serum albumin was used as standard. α-Amylase Purification The supernatant of culture was brought to 70 % ammonium sulfate saturation in an ice bath. The precipitated protein was collected by centrifugation at 3,000×g at 4 °C, dissolved in one to two pellet volumes of phosphate buffer (0.1 M, pH 7.0) then dialyzed overnight at 4 °C against the same buffer. The enzyme solution was concentrated over sucrose bed prior to application to a gel filtration column of Sephadex G-50 (30×2.5 cm) equilibrated

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with Tris–HCl buffer (50 mM, pH 8). The enzyme was eluted with the same buffer; each fraction volume is 2 ml. The active fractions were pooled, concentrated, and applied onto an anion exchange column of DEAE-cellulose (30×2.5 cm) equilibrated with Tris–HCl buffer (50 mM, pH 8). The protein was eluted with a linear gradient of 0.0 to 1.0 M NaCl in the same buffer; each fraction volume is 2 ml. Physicochemical Properties of α-Amylase Enzyme activity at various temperatures and pH was studied by incubating reaction mixtures at different temperatures (30, 40, 50, 60, 70, 80 °C) and Tris–HCl buffer (pH 4, 5, 6, 7, 8, 9). Enzyme stability at various temperatures was also studied by pre-incubating cell-free supernatants for different times at various temperatures. The effect of metal salts (NaCl, CoCl2, and CaCl2) and EDTA on activity was determined by adding different concentrations (1, 5, 10 mM) of each salt to the standard assay. Activities were expressed as a percentage of the maximal activity. The carbohydrate content of the purified enzyme was determined according to the method described by Dubios et al. [12]. Thin Layer Chromatography Products released, as mode of action of purified α-amylase, through starch hydrolysis were separated by ascending TLC using the solvent n-butanol/ethanol/water (5:3:2 v/v). The chromatogram, plate coated with silica gel (Merck, 20×20 cm), was developed, dried, sprayed with a solution of acetone–silver nitrate solution (0.1 ml saturated solution of AgNO3 in 20 ml acetone), and heated at 90 °C for 2 min. Glucose and maltose were used as standards, each at 0.5 % (w/v) concentration. Electrophoresis and Molecular Weight Determination Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) was carried out using Bio-Rad Mini-Protean ΙΙ Cell Gel apparatus at 200 V. Gels were prepared according the method of Laemmli [13] using 10 % concentration of acrylamide. SDS was omitted when the activity was to be retained. The reference pre-stained protein markers (# SM 0671; Fermentas) were used. Protein and glycoprotein bands were visualized by silver stain according to the method of Heukeshoven and Dernick [14] and the method of Moller and Poulsen [15], respectively. Amylase activity of proteins was detected according to Garcia-Gonzalez et al. [16] by incubating the gels at 50 °C for 20 min in 0.2 M phosphate buffer (pH 7.0) containing 2 % starch and then immersing in staining solution (KI 13 g/l and I2 6 g/l). The gel was destained with distilled water. The stain was stable for only a few minutes. DNA Manipulation The genomic DNA was isolated according to Salgado et al. [17] in a 320–480 W microwave oven for 15–20 s. Agarose gel electrophoresis was carried out (Shelton DNA electrophoresis, Model JSB120, UK) in 1× TAE buffer at 120 V. The DNA and PCR products were examined parallel to DNA ladder (#R0491; Fermentas) by 2 % agarose gel. The specific primers used for amplification of α-amylase gene (forward primer 5′ CAATGGTGATCCCAGCA3′ and reverse primer 5′CGCCCATAAGCATAAAGT3′) were designed from two conservative regions chosen from the α-amylase DNA sequence alignment of tvaI, tvaII, and amyTV1 genes from T. vulgaris with accession numbers D13177, D13178, and X69807, respectively. The PCR mixture consisted of 30 pmol of each primer, 1 μg of chromosomal DNA, and 12.5 μl DreamTaq Green PCR Master Mix (K1081; Fermentas) to a final volume of 25 μl in water-free nuclease. The reaction was run in a thermal cycler



(TECHNE model FTC3102D, UK) programmed for 30 cycles as follows: initial denaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s, annealing at 41 °C for 30 s, primer extension at 72 °C for 2 min, and final extension at 72 °C for 10 min. The band of the PCR product was extracted from agarose gel, using Gene JETTM Gel Extraction Kit (K0699; Fermentas), and sequenced (GACC, Germany) using the two previous primers. Statistical Analysis Data were statistically analyzed for variance and the least significant difference (LSD) using one-way analysis of variance (ANOVA) by software system SPSS version 15. Gene alignment and similarity analysis were achieved by the software system Clustal W version 1.82 [18].

Results Purification of α-Amylase Extracellular α-amylase was purified using different steps of concentrations and column chromatography. The first step of α-amylase purification was performed by ammonium sulfate precipitation. The specific activity increased from 1,219.9 U/mg protein to 2,143.8 U/mg protein, with a purification factor of 1.76 (Table 1). After dialysis, the concentrated enzyme was applied to a gel filtration column in which one large peak was detected (Fig. 1a) with an increase of specific activity 127,100.3 U mg−1 protein corresponding to a 104.2-fold increase in purification (Table 1). The active fractions obtained from gel filtration column were further purified using anion exchange chromatography. The most purified and active enzyme was eluted in fractions 75 to 92 by a NaCl linear gradient (0.7–0.9 M) (Fig. 1b) possessing a specific activity of 59,697.3 U mg−1 and representing a purification factor of 48.9 (Table 1). Physicochemical Properties of the Purified α-Amylase Effect of pH The effect of pH on the activity of the purified α-amylase was studied over a range of pH 4 to pH 9 (Fig. 2). The activity increased gradually over a range of pH 4 to pH 7 with the maximum level at pH 6. Seventy-six percent of the relative activity in the range pH (4 to 5) was retained, while 85 % of the activity was retained at pH 6. Above pH 7, the

Table 1 Purification summary of extracellular α-amylase of T. vulgaris using ammonium sulfate precipitation, gel filtration chromatography, and anion exchange chromatography Step

Total activity (U)

Total protein (mg)

Specific activity (U/mg)

% αamylase

Purification fold

Crude supernatant Ammonium sulfate precipitation

124,448.08 49,644.52

102.02 23.16

1,219.89 2,143.83

100.00 39.89

1.00 1.76

Gel filtration chromatography

1,067.48

8.35

127,100.33

14.52

104.19

Anion exchange chromatography

2,929.14

5.40

59,697.26

2.35

48.94


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Fig. 1 Purification of α-amylase of T. vulgaris using gel filtration column containing Sephadex G-50 (a) and anion exchange column containing DEAE-cellulose (b)

activity of the enzyme decreased significantly (LSD at 0.01 level) and retained 58 % and 49 % of the original activity at pH 8 and pH 9, respectively. Effect of Temperature The α-amylase activity increased significantly (LSD at 0.01 level) by 2.5-fold when the temperature was increased from 30 °C until it reached the optimum activity at 50 °C (Fig. 2). When the temperature was raised above 60 °C, the activity decreased significantly retaining 73.1 % and 50 % of the original activity at 70 °C and 80 °C, respectively. The activation energy (E) of the purified enzyme was found to be



Fig. 2 Effect of pH and temperature on purified α-amylase activity. The enzyme activities are represented relative to the maximal values

5,375.8 kJ. The thermal stability of the purified enzyme was investigated over 240 min at a range of temperature from 50 °C to 90 °C (Fig. 3). At the first 30 min, a rapid decrease at the enzyme activity was detected at different tested temperatures. The activity declined rapidly reaching zero value after 240 min at 60 °C while retaining only 33.4 % of the original activity at 50 °C. The purified enzyme has a half-life of 120 min at 50 °C. However, it was still keeping about 45 % of its activity at 90 °C after 60 min. Effect of Metal Ions The purified enzyme was found to be sensitive to different metal ions used, which included NaCl, CoCl2, and CaCl2 (Fig. 4). Both NaCl and CoCl2 affected the purified enzyme similarly; 1 mM concentration did not have any significant effect (LSD at 0.01 level) on the activity. However, increasing the concentration induced a highly significant decrease in the activity retaining only about 43 % of the original activity at 10 mM concentration. Using 1 mM of CaCl2 resulted in a significant inhibition by 36.1 % of the original activity, although this inhibitory effect decreased significantly with increasing the concentration of CaCl2 until the activity reached 96.7 % at 10 mM. Effect of Different Concentrations of EDTA α-Amylase of T. vulgaris was not significantly influenced by 0.01, 0.1, 0.5, or 1 mM EDTA (LSD at 0.01 level) even if 0.5 mM EDTA was found to cause a slight increase in the enzyme activity up to 123.8 %, while 0.001, 5, and 10 mM EDTA declined the enzyme activity significantly by 48.8 %, 82.3 %, and 94.7 %, respectively (Fig. 5). Carbohydrate Content of the Purified α-Amylase α-Amylase of T. vulgaris has high carbohydrate content (341.26 μg/ml) compared with its protein content (18.67 μg/ml), showing a high degree of glycosylation that reached 94.81 %.


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Fig. 3 Effect of temperature on the stability of the purified α-amylase of T. vulgaris. The enzyme activities are represented relative to the maximal value

Mode of Action of the Purified α-Amylase The TLC showed that the end products of the starch hydrolysis by α-amylase were maltooligosaccharides (Fig. 6). Electrophoresis and Molecular Weight Determination Silver staining could not detect any bands corresponding to the purified proteins resulting from gel filtration and ion exchange

Fig. 4 Effect of different concentration of different metal ions on the purified α-amylase activity. The enzyme activities are represented relative to the control activity



Fig. 5 Effect of different concentration of EDTA on the purified α-amylase activity. The enzyme activities are represented relative to the control activity

columns compared with the crude filtrates (data not shown). Applying the silver staining of glycoprotein, for the purified proteins from gel filtration and ion exchange columns, two bands of approximate molecular weight of 135 and 145 kDa were detected. They were found to coincide with that of the active amylase bands determined by activity staining (Fig. 7). Putative α-Amylase Gene Amplification and Sequencing The PCR product examination on agarose gel electrophoresis revealed a single band in the expected size of 1,100–1,200 bp. DNA sequencing of the partial amplified putative α-amylase gene from T. vulgaris (putative amyTVE) gave 1,089 bp which revealed an open reading frame of 363 amino acids (GenBank accession no. HE599529). The putative AmyTVE amino acid alignment showed 56.2 % similarity with other thermoactinomycetes α-amylases (Fig. 8). The alignment with α-amylase (AmyTV1) from T. vulgaris 94-2A and α-amylases (TvaI and TvaII) from T. vulgaris R-47 revealed two conservative regions (GxRxDxx and xxxGE) for active sites II and III, respectively (Fig. 9).

Discussion Classical purification methods of α-amylases involve separation of the culture from the fermentation broth and selective concentration by precipitation using ammonium sulfate or organic solvents. The crude enzyme is then subjected to chromatography including ionexchange and gel filtration chromatography, which are usually combined [19]. Although the precipitation step caused a reduction in the yield of the enzyme, this corresponded to a purification factor of 1.76. The gel filtration chromatography step increased the specific activity representing a purification factor of 104.19. In this study, even though α-amylase was detected electrophoretically to have 135 and 145 kDa, it was eluted and successfully


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Fig. 6 Thin layer chromatography (TLC) of products of starch hydrolysis by α-amylase of T. vulgaris (2). Standards in the order of migration, starting at the origin slowest to fastest moving components, are maltose and glucose (1)

purified using a Sephadex G-50 matrix. One possibility is that α-amylase penetration of the matrix would be facilitated by its shape. However, the crystal structure of the enzyme is



Fig. 7 SDS–PAGE glycoprotein staining of partially purified α-amylase after gel filtration (a), purified αamylase after anion exchange (b), and zymogram of purified α-amylase (c)

necessary to solve that mystery. This diffusion of the enzyme through the small pores of Sephadex despite its high molecular weight would be advantageous for industrial applications. Unfortunately, processing of the enzyme through the ion exchange DEAE-cellulose column resulted in a highly reduced enzyme activity that the purification fold was decreased. In spite of that, it exhibited two highly pure protein bands on SDS–PAGE with a purification fold of 48.94. In this step, the eluting solution may interact with the enzyme leading to different conformational states, which may result in different aggregation behavior that reduced the activity. Protein aggregation may be induced under mild purification conditions

Fig. 8 Relatedness dendrogram of putative AmyTVE, AmyTV, TvaI, and TvaII α-amylases


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Fig. 9 Amino acid sequence alignment of two short conserved sequence segment II and III of putative AmyTVE compared with AmyTV, TvaI, and TvaII α-amylases

[20, 21]. In reality, many complex relationships could be explained between protein aggregation and the solution ionic strength [22] which may be dependent on the solution pH [23]. Furthermore, the pure enzyme characters were found to differ slightly from that of the crude enzyme [6]. Since the solution pH dictates the type and distribution of surface charges on proteins, it is expected to play a critical role in controlling the enzyme activity and stability. It is worth noting that the α-amylase active site consists of a large number of charged groups, among which are the nucleophilic aspartate and the catalytic hydrogen donor glutamic acid [24–27]. As a result, α-amylase catalysis is thought to be limited by the protonation of the nucleophile at low pH and by deprotonation of the hydrogen donor at high pH [28]. In comparison to other published data, this α-amylase showed wider pH range than other Thermoactinomyces α-amylases [29–31], which makes it an industrially useful enzyme. Comparing with other α-amylases from T. vulgaris 94-2A, T. vulgaris R-47, and Thermoactinomyces thalpophilus, they have optimum temperatures of 62.5 °C, 70 °C, and 90 °C, respectively [29–31]; our pure enzyme is distinguished by being an intermediate temperature stable (ITS) α-amylase. This feature was found by Olesen [32] to render the enzyme to be useful for baking industry as it is favorable to avoid stickiness in bread. It is worth noting that α-amylase with “ITS” property has been reported from only a few microorganisms [4]. The purified α-amylase has a significantly reduced thermal stability compared to its crude form [6]. This may be due to fractionation of such chaperon heat-shock proteins during purification and/or removal of some metabolites that would disrupt the heat protection they afford to the enzyme. In support, the protective and stabilizing effect of cosolvents such as sugars and polyhydric alcohols against thermal denaturation and inactivation of various enzymes has been extensively described [33–40]. However, the enzyme has a half-life time of about 1 h at 90 °C. This low rate of irreversible denaturation could be due to the large activation energy value [41]. Co2+, Na+, and Ca2+ have an inhibitory effect on the enzyme, in contrast to T. vulgaris R47 pure α-amylase that is strongly stabilized by Ca2+ [29]. All known α-amylases, with a few exceptions, contain a conserved Ca2+ binding site [42, 43]. In the manufacture of fructose syrup, Ca2+ ions inhibit the glucose isomerase enzyme used in the final step of the process [2] and may lead to the formation of inorganic precipitates which have deleterious effects on fermentation and downstream processing [44]. Because removal of these metal ions is both cost and time consuming to the overall industrial process [44], the use of stable and functional α-amylases in the absence of Ca2+ ions at high temperatures would be highly favored. Also, Cl− seems to be a stabilizer since it reverses the Ca2+ inhibitory effect. Chloride ions have been found mainly in the active site of mammalian α-amylases, which have been shown to enhance the catalytic efficiency of the enzyme [43]. In addition, αamylase produced by Bacillus thermooleovorans was found to contain a chloride ion in their active site [45], which enhances the catalytic efficiency of the enzyme, presumably by elevating the pKa of the hydrogen-donating residue in the active site [43]. However, in this



study; chloride does not reverse the Na+ effect. It could be concluded that the mechanisms of inhibition by Ca2+ and Na+ are different. The irreversible inhibition effect of sodium on the pure enzyme, which is different from that in the crude form [6], may reflect a high sodium accumulation during the ion exchange chromatography. This may be the cause of the activity reduction. Both positive and negative ions in the eluting solution can potentially bind to or interact electrostatically with proteins. Such interactions can lead to altered charge–charge interactions and even different conformational states, which may result in different aggregation behaviors [20, 21]. Therefore, to further improve the purification process, sodium ions should be removed or eluted by a gradient of other salt solution. In support to the previous hypothesis, EDTA at 0.5 mM concentration was found to increase the enzyme activity. This may be due to the removing of sodium ions through chelating. At lower concentration, the poorly populated protein folding/unfolding intermediates are precursors of the aggregation process. But completely folded or unfolded proteins, in contrast, do not aggregate easily since the hydrophobic side chains are either mostly buried out of contact with water or randomly scattered [21]. Since the enzyme product was separated as maltooligosaccharides without any maltose, it suggested an endo-mode of action for the amylase of T. vulgaris which may be of the multichain attack mode [46]. On the other hand, α-amylase of T. vulgaris 94-2A was found to produce maltose and maltotriose [30]. Glycosylation is one of the major post-translation modifications that can affect a variety of enzyme functions [47, 48]. The α-amylase of this investigation has a high degree of glycosylation (94.8 %). In comparison, a carbohydrate content as high as 56 % has been reported for α-amylase of Schwanniomyces castelii [49] whereas this is about 10 % for other α-amylases [50–54]. Coomassie brilliant blue and silver stains are far less sensitive when used for detection of highly glycosylated protein leading to weak staining or even failure of detection. This is presumably owing to steric interference by the carbohydrates with the binding of silver ions [15]. Therefore, glycoproteins can be detected by initial oxidation of carbohydrates by periodic acid and subsequent staining with cationic dyes such as alcian blue [55]. It is worth noting that SDS–PAGE indicated a high purity of the enzyme as the silver stain of protein, despite its sensitivity, could not detect any protein band. But only silver stain of glycoprotein could detect only two broad pure bands that correspond to the active bands of the α-amylase. These broad, fuzzy bands were attributed to the variable number of negatively charged carbohydrates which can seriously reduce the resolving power of the SDS–PAGE technique [56]. The present result revealed a highly molecular weight α-amylase isozymes 135 and 145 kDa, part of which due to the high glycosylation [7]. Molecular weights of αamylases vary from about 10 to 210 kDa. The lowest value of 10 kDa for Bacillus caldolyticus [57] and the highest value of 210 kDa for Chloroflexus aurantiacus [58] were reported. Molecular weights of microbial α-amylases are usually 50–60 kDa as shown directly by analysis of cloned α-amylase genes and deduced amino acid sequences [53]. T. vulgaris R-47 was reported to produce two α-amylases, TVAI and TVAII, with molecular weights of 71 kDa and 67.5 kDa, respectively [59]. Also, α-amylase of T. vulganis 94-2A (AmyTV1) is a protein of 53 kDa, and smaller peptides of 33 and 18 kDa have been shown to be products of limited AmyTV1 proteolysis [30]. The DNA and amino acid sequence similarities showed that the recently identified putative AmyTVE is more related to AmyTV1 from T. vulgaris 94-2A than TVAI and TVAII from T. vulgaris R-47. A possible distant sequence relationship between the putative AmyTVE and thermostable α-amylases (TVAI and TVAII) may be retained to structure–


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specificity relationships [60]. Specificity studies on TVAI and TVAII enzymes showed that they can hydrolyze cyclodextrins and α-1,6 linkage in small oligosaccharides in addition to the α-1,4 linkages [59], whereas putative AmyTVE was confirmed to hydrolyze only α-1,4 linkages by the TLC analysis of its sugar products. In addition, this suggestion could be confirmed by its relatively higher similarity with AmyTV1 whose activity is only against α1,4 bonds [30]. The distinction between the putative AmyTVE, TVAI, TVAII, and AmyTV1 led us to confirm the suggestion of either variability of amylase genes among the T. vulgaris strains [30] or a still uncertain heterogenicity of the genus Thermoactinomyces [61–64]. The sequence alignment also revealed two short conserved amino acid sequences which represent the conserved sequence segment II and III in α-amylases [60]. These were appeared to generally constitute subsites +1 and +2 [27, 65–67] that are essential for catalysis [68, 69]. The glutamic acid residue in segment III was believed to be the proton donor, while the aspartic acid in segment II was thought to act as the nucleophiles in the displacement catalytic steps [27]. In conclusion, the present study indicated a novel active highly molecular weight, highly mobile, intermediate thermostable, with wide pH range and calcium-independent α-amylase of T. vulgaris which could be of importance for different industrial applications.

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