Molecular Cloning and Biochemical Characterization of a novel thermostable α-amylase of Bacillus subtilis MDC 3500 isolated from acidic soils in Armenia

doi:10.21203/rs.3.rs-4103518/v1

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Molecular Cloning and Biochemical Characterization of a novel thermostable α-amylase of Bacillus subtilis MDC 3500 isolated from acidic soils in Armenia

https://doi.org/10.21203/rs.3.rs-4103518/v1

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Amylases are one of the most important industrial enzymes, accounting for 30% of the world's production of enzymes. The quest for novel recombinant α-amylases with enhanced traits remains a pressing challenge, presenting continual relevance in biotechnological sectors. Bacillus subtilis strain MDC 3500 was isolated in acidic soils (pH 3.5-4.0) of Armenia. In this study, the α-amylase gene of Bacillus subtilis MDC 3500 (AmyBS) was cloned by the golden-gate cloning technique followed by intracellular expression in Escherichia coli cells. Phylogenetic analysis revealed a close relationship between AmyBS and α-amylases of Bacillus subtilis A28, exhibiting 97.7% homology. AmyBS was expressed and purified to homogeneity using a two-step purification process involving immobilized metal affinity chromatography and size exclusion chromatography. The temperature and pH optimum, thermal stability, and several other catalytic characteristics of AmyBS were studied. The enzyme exhibits the following order of starch substrate preference: potato > wheat > corn > rice. AmyBS also exhibits specificity for amylose, amylopectin, γ-cyclodextrin, and β-cyclodextrin in decreasing order. The hydrolytic products of potato, corn, or rice starches mainly lead to the accumulation of glucose, maltose, and, to a lesser extent, maltotriose in the reaction medium.

Bacillus subtilis MDC 3500

Acidic Soils

α-Amylase

Characterization

Golden Gate cloning

α-Amylases (E.C. 3.2.1.1.) are starch-degrading enzymes that hydrolyze the internal α-1,4-O-glycosidic bonds of polysaccharides, preserving the α-anomeric configuration of the products. Most α-amylases are metalloenzymes that require calcium (Ca²⁺) ions for their activity, structural integrity, and stability. They belong to the glycoside hydrolase enzyme family 13 (GH-13) based on amino acid sequence similarity [1]. Amylases are one of the most important industrial enzymes, widely used for converting starch into sugar syrups abito produce cyclodextrin for the pharmaceutical industry. They account for 30% of the world's production of enzymes [2].

The α-amylase GH-13 family cosist of the GH-H clan of glycoside hydrolases (this clan also includes the GH-70 and GH-77 families), which is the largest family of transferases and isomerases, represented by more than 30 different enzyme forms(Dhanya Gangadharan et al. 2006) [3]. These enzymes include endoamylases, exoamylases, debranching enzymes, and transferases [3].

The α-amylase family, or the GH-H clan of the glycoside hydrolases, is the largest family of glycoside hydrolases, transferases, and isomerases, represented by more than 30 different enzymes [3].

In many works, conservative sites of the primary structure of α-amylases, domain and spatial structures, and catalysis mechanisms were studied in detail [4].

α-Amylases are ubiquitous and produced by plants, animals, and microbes, which play a dominant role in carbohydrate metabolism. Amylases of plant and microbial origin have been used as food additives for centuries. Barley amylases have been used in beer production. Fungal amylases have been widely used as target foods. Despite the widespread use, mainly amylases of microbial origin (bacterial and fungal) are used in industry (B. subtilis, B. stearothermophilus, B. Licheniformis, and B. amyloliquefaciens) due to their cheapness, ease of production, and the possibility of modifying and optimizing the production process [5, 6].

In the processes of application of α-amylases, the optimal temperature and pH of their activity, as well as heat stability, are essential. According to these parameters, searching for and describing new α-amylases are among the eternal biotechnological problems [5, 7].

The effects of Ca²⁺, Mg²⁺, Mn²⁺, Zn²⁺, Fe²⁺, and other metal ions on amylase activity have been studied in many works [5].

Substrate specificity of amylase against soluble starch, amylose, amylopectin, glycogen, maltodextrin, cyclodextrins, and other substrates has been studied in many modern works [8, 9]. The search for new α-amylases that hydrolyze raw starch (the technologies with their usage lack the energy-intensive stage of starch gelatinization) is one of the urgent problems of modern biotechnology [10, 11].

The demand for α-amylases with different physiological and biochemical characteristics in various productions is conditioned by the use of recombinant technologies [12–14] and the targeted search for enzymes with new features using enzymatic engineering [15, 16]. Using point mutagenesis, in the result of deletion of amino acids Arg¹⁷⁹ and Gly¹⁸⁰ of α-amylase of Bacillus stearothermophilus, a recombinant enzyme with improved characteristics was obtained and described (high heat resistance, the ability to work at low pH, low Ca²⁺ requirement). Due to the optimization of the signal peptide of α-amylase of B. stearothermophilus and overexpression of the respective chaperone, it was possible to achieve the overexpression of the secreted enzyme (9200 units/ml) [17].

Thus, the preparation and characterization of producer strains of new recombinant alpha-amylases with improved characteristics and corresponding enzymatic forms, which are widely used in the processing of starch-containing raw materials, is an urgent task, and the efforts made in this direction remain in demand by biotechnological industries.

The aim of this work was the molecular cloning of the α-amylase gene of the B. subtilis 3500 strain in E. coli vector pET28gg-LacZ for obtaining an expressing His-tagged recombinant enzyme and its subsequent characterization.

Reagents

Phusion® DNA Polymerase, BsaI restriction enzyme, and T4 DNA ligase were purchased from New England Biolabs (Hitchin, UK). Isopropyl β-D-1-thiogalactopyranoside (IPTG) was purchased from Merck, UK. The molecular weight marker for SDS–PAGE was purchased from Thermo Fisher Scientific (Cramlington, UK). 3,5-dinitrosalicylic acid and starch samples were purchased from Sigma-Aldrich, US.

Bacterial strains and vectors

The Bacillus subtilis MDC3500 strain, used as the source of the α-amylase gene, was isolated from a soil sample with pH 3.5 and is currently maintained at the Microbial Depository Center, SPC “Armbiotechnology,” NAS RA. The strain's 16S rRNA gene was sequenced and accessible in GenBank under the accession number MT534524.1. For primer design, the genome sequence of Bacillus sp. KBS0812 strain, displaying 99.8% identity to the 16S rRNA sequence of Bacillus subtilis MDC3500, was used as a template. In the cloning and expression processes, E. coli Top10 and E. coli BL21(DE3) were used as cloning and expression hosts, respectively. Golden Gate cloning and expression were realized using a modified pET28 plasmid, kindly provided by Dr. Jon Marles-Wright. Cultivation of the strains was carried out aerobically at 37°C in Lauria–Bertani (LB) medium, comprising 1% w/v peptone, 0.5% w/v yeast extract, and 1% w/v NaCl, adjusted to pH 7.0. This medium served as the optimal environment for the growth of the strains during cloning and expression procedures.

Cloning and sequence analyses

Benchling software was used for specific primer design, in silico PCR, and assembly experiments. For cloning, AmyBS gene was amplified using Amy-F (5' GACGGTCTCTAATGGAAACGGCGAACAAATCGAATGAGC 3') and Amy-R ( 5'GTCGGTCTCTACCTATGGGGAAGAGAACCGCTTAAGC 3') primers (primer extending sequences are indicated in bold) within the following PCR conditions: 95°C for 1 min followed by 30 cycles of 95°C for 30 s, 65°C for 30 s and 72°C for 1 min, followed by a final elongation at 72°C for 10 min, using Bacillus subtilis MDC 3500 genome DNA as a template and after examination by electrophoresis, the amplified product was purified with the QIAquick PCR Purification Kit (Qiagen), then assembled via one-pot Golden Gate cloning [18] and CIDAR MoClo system [19] and used to transform E. coli TOP10 cells. Recombinant plasmid DNA was extracted from insert-positive clones and, after sequence confirmation, used to transform E. coli BL21(DE3) cells.

Sequence homology analysis of the AmyBS gene was performed using the BLAST program [20] Protein sequence alignments were performed by PRABI (Pôle Rhône Alpin de Bioinformatique) Institutes Multalin software [21] and figures prepared with ESPript [22]. The phylogenetic tree was constructed using MEGA11, calculated the neighbor joining method [23]. The evolutionary history of the AmyBS was inferred using the Maximum Likelihood method and JTT matrix-based model [24]. The structure of AmyBS was generated using the Phyre2 server [25] visualized by Mol*3D viewer [26]. The theoretical molecular weight (MW) and isoelectric point (pI) were estimated using the ExPASy, ProtParam tool Compute MW/pI [27].

Protein expression and purification

For protein expression, E. coli BL21(DE3) harboring the plasmids pET28GGLacZ::AmyBS was grown in LB media (35 µg/mL kanamycin) at 37°C and 160 rpm. Gene expression was induced at OD₆₀₀ = 0.5–0.6 by adding IPTG to a final concentration of 1 mM. Cells were harvested by centrifugation at 9000 × g at 4°C for 20 min. The harvested cells were resuspended in 10 x w/v Buffer HisA (50 mM imidazole, 500 mM NaCl, 50 mM Tris-HCl, pH 8.0) and subsequently sonicated on ice for 5 minutes with 30 s on/off cycles at 60 watts power output. The lysate was cleared by centrifugation at 35,000 × g and filtered with a 0.45 µm syringe filter.

The cell-free extract was applied to a 5 ml HisTrap FF column (GE Healthcare), and unbound proteins were washed off with 10 column volumes of Buffer HisA (50 mM Tris-HCl, pH 8.0, 500 mM NaCl, 50 mM imidazole). A step-gradient of 50% and 100% Buffer HisB (50 mM Tris-HCl, pH 8.0, 500 mM NaCl, 500 mM imidazole) was used to elute His-tagged proteins. Fractions of His-trap eluent containing the protein of interest were pooled and concentrated by Vivaspin Turbo (Sartorius, 10 kDa MWCO) centrifugation devices at 4,000 × g, 18˚C. The concentrated protein was then subjected to size-exclusion chromatography using an S200 16/60 column (Cytiva), equilibrated with Buffer GF (50 mM Tris-HCl, pH 8.0, 150 mM NaCl).

Purified AmyBS was concentrated and analyzed by SDS–PAGE (12.5%) [28] after incubating the sample for 5 min at 95°C temperature in the presence of 5% β-mercaptoethanol and 1% SDS. The Fermentas pre-stained PageRuler was used as a protein molecular weight marker for SDS-PAGE. Gels were stained with Coomassie brilliant blue for the visualization of protein bands.

Protein concentration was determined according to Bradford [29], with bovine serum albumin as standard.

Characterization of AmyBS

Unless otherwise noted, the standard assay was carried out at 65°C for 10 min in 200 µL reaction mixture using 1% (w/v) soluble starch from potato (Sigma-Aldrich) as substrate in 50 mM sodium phosphate buffer pH 6.0 (reaction mixture) and a necessary amount of enzyme preparation. For each assay, the reaction mixture was incubated at the desired temperature for 10 minutes, after adding an amount of enzyme required. After a 10-min reaction, the sample was placed on ice to stop the reaction. Additionally, blanks without enzyme were performed by default for all measurement series. The hydrolytic activities of the purified enzymes were detected by measuring the reducing sugars with 3,5-dinitrosalicylic acid (DNS), according to Miller [30], with glucose as standard. In brief, after the enzyme reaction, 200 µL reaction mixture was mixed with 500 µL H₂O and 500 µL DNS reagent (1% (w/v) DNS, 30% (w/v) potassium sodium tartrate, 0.4 M NaOH) and were incubated for 5 min at 100°C. Samples were cooled on ice to room temperature and absorption was measured at 546 nm. All measurements were done in duplicate. One unit of enzyme activity was defined as the amount of enzyme required to release 1 µmol of reducing sugars per minute.

The influence of temperature on enzyme activity was examined by performing the standard assay at different temperatures varying from 35 to 80°C. To investigate the temperature stability, the enzyme samples were pre-incubated in the same temperature range for 15 minutes following the incubation on ice. Thermostability experiments were also performed in the presence of 5 mM CaCl₂ to investigate the thermostabilizing effect of Ca²⁺ ions. Residual activities were measured according to the standard assay.

To assess the impact of pH on enzyme activity, a standard assay was conducted using Britton-Robinson buffer (50 mM) within a pH range of 3.5-8.0 in the reaction mixture [31]. The pH-dependent thermostability of the enzyme was investigated by incubating enzyme samples at 65°C for 15 minutes across a range of pH values. Subsequently, the residual activity was measured under standard assay conditions.

The influences of metal ions on enzyme activity were analyzed by using a standard assay after incubating the enzyme in the presence of 5 mM FeCl₂, BaSO₄, CdCl₂, CuCl₂, ZnCl₂, MgCl₂, CaCl₂, MnCl₂, CoCl₂, NiCl₂, KCl, or NaCl for one hour at room temperature. The influences of detergents (1%) such as SDS, Triton X-100, Triton X-405, Tween 80, reducing agents (5mM) such as dithiothreitol (DTT) and β-mercaptoethanol (ME), 5 mM of 5,5՛-dithiobis-(2-nitrobenzoic acid) (DTNB), and 10 mM of EDTA, were examined by the standard assay procedure followed by one-hour incubation at room temperature. A blank experiment was performed for each metal and additive.

For analyzing the substrate spectrum, the specific activity of AmyBS was measured at 65°C, pH 6.0 for 10 min using different substrates. The tested substrates were soluble starch, potato starch, Zukovski corn starch, rice starch, potato starch, amylose, amylopectin from potato, α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin. All substrates were used at a final concentration of 1% (w/v) in phosphate buffer (50 mM, pH 6.0). A blank experiment was conducted for each substrate.

Determination of the Hydrolysis Products

For determination of the hydrolysis products, 0.5% (w/v) substrate was incubated with AmyBS in standard reaction mixtures at 60°C for 18 h. After the heat inactivation of the enzyme in a boiling bath at 96°C for 10 min, samples were centrifuged (20.000 × g, 10 min), and the supernatant was filtered through a 0.22 µm membrane filter unit. Hydrolysis products were analyzed by high-performance liquid chromatography (HPLC) under the following conditions: Aminex HPx-42A column (Bio-Rad), 80°C, 0.6 mL/min flow rate, water as mobile phase, HPLC 1260 Infinity II LC System (Agilent Technologies) with RI detector. maltohexose, maltopentose, maltotetrose, maltotriose, maltos, and glucose were used as standards.

Statistical analysis

All the results were reported as mean values of 3 measurements. Calculations were done using the Microsoft Ofce Excel 2016 software package. Results with p < 0.05 were considered significant.

Nucleotide sequence accession number

The sequence of AmyBS was deposited in GenBank (accession number UUZ04253.1).

Bacillus subtillis 3500 strain was isolated from the soil samples with a pH of 3.5. As an acidic α-amylase producing strain, it has been kept in the Microbial Depository Centre (MDC) of the SPC “Armbiotechnology” NAS RA under the name Bacillus acidophilus MDC 3500. The initial characteristics of the α-amylase exhibited great promise, prompting the decision to clone and characterize the enzyme. Through 16S rRNA gene sequencing, the strain demonstrated a 99% identity to Bacillus sp. KBS0812 (GenBank: CP041757.1), which genome served as a template for primer design for α-amylase encoding gene cloning. The AmyS gene (GenBank: UUZ04253.1) has 1886 bp size, encoding the enzyme (AmyBS) that consists of 658 amino acids. Domain prediction has revealed that the α-amylase comprises several functional domains. These include a signal peptide that undergoes cleavage between the 31st and 32nd amino acids. Additionally, it possesses an alpha-amylase (Aamy) catalytic domain, which is typical for maltogenic α-amylases, belonging to family 13 of the glycosyl hydrolases. Enzymes having this domain catalyze the hydrolysis of (1–4)-α-D-glucosidic linkages in polysaccharides, removing successive α-maltose residues from the non-reducing ends of chains during the conversion of starch to maltose. Furthermore, AmyBS has also an Amy_C domain, a C-terminal ß-sheet domain that appears to show some variability in sequence and length between amylases (domain C) [32] and CBM26, a carbohydrate-binding module (Supplementary Fig. 1). Based on the amino acid sequence, a theoretical molecular mass of 69.01 kDa and an isoelectric point (pI) of 5.52 were calculated for the AmyBS (without signal peptide).

The protein sequence of the recombinant AmyBS exhibits a remarkable 99–100% identity with α-amylases from Bacillus subtilis, as recorded in the NCBI database. Interestingly, none of these enzymes has been characterized to date. Notably, AmyBS displays less than 98% identity with characterized enzymes. Specifically, it shares 97.7% identity with the α-amylase from Bacillus subtilis A28 (GenBank: AFQ31615.1), 97.6% identity with the α-amylase from Bacillus subtilis DR8806 (GenBank: AGW23675.1), and 97.4% identity with the α-amylase from Bacillus subtilis N7 (AMY_BACSU UniProtKB/Swiss-Prot: P00691) in all cases with query coverage of 100%. Furthermore, it demonstrates 91.4% identity with the α-amylase from Bacillus sp. KR-8104 (GenBank: ACD93218.3). Detailed alignment results with these mentioned enzymes are presented in Fig. 1.

Phylogenetic analysis (Fig. 2) indicated that AmyBS forms a cluster with α-amylase of Bacillus subtilis A28, which is in good agreement with the alignment results (the highest 97.7% identity).

To investigate the biochemical properties of AmyBS, a C-terminally hexahistidine-tagged recombinant version of the enzyme was produced in E. coli BL21(DE3), and the protein was then purified through a two-step purification process using immobilized metal affinity chromatography (Fig. 3a) and size exclusion chromatography (Fig. 3b). After purification, the specific activity of AmyBS was increased more than 20 times, reaching 1280 U/mg (Supplementary Table 1). SDS-PAGE analysis revealed a predominant band at approximately 69 kDa, which aligns with the calculated molecular weight of the protein (69.01 kDa ) (Fig. 3c).

To evaluate the influence of temperature on the activity profile of AmyBS, the purified enzyme activity was assayed at temperatures ranging from 40 to 80°C during the reaction times of 10 minutes. The AmyBS α-amylase showed highest enzymatic activity in the temperature range of 55°C to 65°C with a temperature optimum at 65°C under the tested conditions (Fig. 4a). However, the activity decreased dramatically above 65°C. The thermostability of AmyBS was assessed by incubating the enzyme at various temperatures for 15 minutes and measuring the residual activity at 65°C. The enzyme exhibited no significant reduction in activity up to 40°C, with a 50% loss of activity observed at 65°C (Fig. 4b blue line). According to existing literature, calcium ions are reported to positively impact α-amylase activity by imparting a thermostabilizing effect [33, 34]. However, in our recorded observations, we did not identify a significant difference in thermostability when 5 mM of CaCl₂ was present during the incubation of enzyme preparations at different temperatures (Fig. 4b green line).

To examine of the impact of pH on the activity profile of AmyBS, the purified enzyme activity was assayed at pH values ranging from 3.5 to 7.5 at 65°C with reaction times of 10 minutes. Normalized reaction progress data showed an optimum pH of 6.0 for AmyBS under the conditions tested (Fig. 4c). Finally, the determining the thermostability of AmyBS as a function of pH revealed an optimum in the pH range of 5.5–7 with a maximum at pH 6 (Fig. 4d).

The purified AmyBS enzyme was incubated in a phosphate buffer with the addition of various cations, chelators, and reducing agents. The effects of various metal ions at 5 mM concentration are shown in Fig. 5a. Fe²⁺ and Cu²⁺ almost completely inhibited the α-amylase activity. Similar results have been obtained for α-amylase of Bacillus subtilis A28, which was also sensitive against mentioned metal ions [35] Cu²⁺ in 5 mM concentration shows strong inhibition activity for Bacillus subtilis DR8806 α-amylase [36]. On the other hand, α-amylase of B. subtilis S8–18 was not inhibited by Fe²⁺ (at the concentration of 5mM) [37]. Cd²⁺ and Zn²⁺ sowed an inhibition effect with 12% and 15% remaining activity, respectively. Co²⁺ and Ni²⁺ ions also inhibited AmyBS activity, but to a lesser extent, with remaining activities varying between 55% and 67%, respectively. However, no considerable effect was seen upon treatment of AmyBS with Ca²⁺, Na⁺, Mg²⁺, Mn²⁺.

The enzyme was found to be highly stable against EDTA by retaining more than 91% activity at 10 mM concentrations, which indicates that AmyBS α-amylase is resistant to chelating agents. Besides, the AmyBS showed stability towards reducing agents such as β-mercaptoethanol (ME) and DTT, as well as the sulfhydryl reagent DTNB, by retaining more than 83% of initial activity. The inhibitory effect of different detergents: 1% solutions of SDS, Triton X-405, Triton X-100, and Tween 80 against the activity of AmyBS was tested (Fig. 5b).

SDS exerted a robust inhibitory effect, causing over 90% inhibition of the enzyme. Triton X-405 and Tween 80 exhibited minor inhibitory effects, with the enzyme retaining 75% and 88% of its residual activity, respectively. Notably, Triton X-100 was not inhibitory under the conditions it used.

To comprehensively understand AmyBS's substrate specificity, we investigated its activity against various substrates, as outlined in Table 1. The enzyme exhibited its highest activity (1263.8 ± 15.8–1063.3 ± 11.4 U/mg) with native and treated (native potato starch and Zukovski soluble potato starch) forms. AmyBS α-amylase activity towards wheat, corn, and rice starches yielded 908.8 ± 8.9 U/mg, 824.4 ± 16.8 U/mg, and 639.2 ± 26.9 U/mg, respectively. Notably, the activity against amylose and amylopectin did not exceed 20% of its maximal activity. Among cyclodextrins, γ-cyclodextrin emerged as a superior substrate compared to the other two. AmyBS demonstrated only minimal activity (1.1 ± 0.6) towards α-cyclodextrin.

Table 1

Substrate specificity of AmyBS
Substrates	Specific activity, U/mg
Potato starch Zukovski	1263.8 ± 15.8
Soluble potato starch	1178.4 ± 34.9
Native Potato starch	1063.3 ± 11.4
Native wheat starch	908.8 ± 8.9
Native corn starch	824.4 ± 16.8
Native rice starch	639.2 ± 26.9
Amylose	222.1 ± 21.4
Amylopectin	187.0 ± 17.8
γ- cyclodextrin	107.2 ± 2.1
ß- cyclodextrin	67.0 ± 16.2
α- cyclodextrin	1.1 ± 0.6

Substrate degradation pattern of AmyBS

The hydrolysis products of starches were investigated by HPLC analysis. Hydrolysis of all potato (Fig. 6a), corn (Fig. 6b), and rice (Fig. 6c) starches mainly resulted in the appearance of glucose (DP1) and maltose (DP2) and to a lesser extent, maltotriose (DP3) and higher oligosaccharides in a very low amount. Thus, AmyBS can hydrolyze α-glycosidic bonds on linear and branched carbohydrate polymers.

In this study, we have presented the production and characterization of a recombinant α-amylase derived from Bacillus subtilis 3500 (AmyBS). The ORF of AmyBS encodes a protein comprised of 659 amino acids, with a predicted molecular mass of 72.45 kDa. The signal peptide of the translated protein was excised during the cloning process, resulting in a 69.01 kDa protein, which was successfully expressed within the Escherichia coli expression system. According to domain prediction results, the enzyme exhibits a C-terminal domain, which was predicted as Carbohydrate- Binding Module 26 (CMB26), comprising 95 amino acids (GYQNPNHWSQVNAYIYKHDGGQAIELTGSWPGKPMTKNADGIYTLTLPADTDTTNAKVIFNNGSAQVPGQNQPGFDYVQNGLYNDSGLSGSLPHR). CMB26 has a calculated molecular weight of 10.28 kDa, which is banded to the Aamy_C domain with an interdomain amino acid strain (Supplementary Fig. 1). The wild-type extracellular α-amylase of Bacillus subtilis 3500 had been previously purified from the culture supernatant. The enzyme revealed a molecular weight of 57 kDa (Supplementary Fig. 2). Comparing these outcomes, we deduce that the wild-type enzyme undergoes the loss of N-terminal signal peptide and C-terminal fragment, resulting in an enzyme with a 57 kDa molecular weight. This observation aligns with the findings of Salimi and coauthors [38]. By comparing the wild-type α-amylase and its cloned variants (with and without C-terminal domain), they have concluded that the C-terminal segment of the enzyme is cleaved during the secretion process. They also suggested that the C-terminal portion lacks additional functionality and does not influence on the enzyme's characteristics.

However, our results present a contrasting perspective. The wild-type amylase exhibits a 1.5-fold higher specific activity towards native potato starch compared to the cloned version (Supplementary Table 2). Conversely, it displays lower hydrolytic activity toward corn starch than the cloned counterpart. In contrast, the C-terminally truncated enzyme from Bacillus stearothermophilus was more than two times less active and less thermostabile [39], and the truncation of the C-terminus did not affect the α-amylases activity from Bacillus subtilis X-23 [40]. Different functions, such as binding to raw starch [34, 41] and thermal stability [34, 39], have been suggested for the C-terminal part of the enzyme. Thus, we concluded that C-terminal truncation did not affect the thermal stability of the enzyme (Supplementary Fig. 3). However, a notable disparity was observed in the temperature-pH profile of enzyme activity. The wild-type enzyme showed a broad spectrum of temperature and pH profiles, while the cloned version exhibited a narrower range. Specifically, the wild-type enzyme displayed 60% of its maximal activity at pH 4.5, whereas the cloned version exhibited 60% (Fig. 4c and Supplementary Fig. 3c). There are also differences in affinity toward different substrates (Table 1 and Supplementary Table 2). These variations in enzyme activity between the wild-type amylase from Bacillus subtilis 3500 and the cloned version expressed in the E. coli expression system could also be attributed to differences in protein conformation arising from the distinct host environments. Indeed, these two hosts offer different cellular milieus with unique protein folding machinery and post-translational modification processes, which may influence the assembly of protein conformation, leading to differences in the activity of the α-amylase [42].

It's worth highlighting that the full-length α-amylase gene of B. subtilis A28 was successfully cloned and expressed in the E. coli JM109 strain. The authors observed amylase activity in the supernatant, indicating that the E. coli secretory system likely secreted the enzyme without the lossing either the signal peptide or the C-terminal region [35]. The findings from another study detailing the cloning of the α-amylase gene from the B. subtilis DR8806 strain and its subsequent expression in the E. coli BL21(DE3) have demonstrated an interesting outcome. Despite cloning the full length of the α-amylase gene, having both the signal peptide and the C-terminal region, the expressed amylase activity was detected within the cell rather than being secreted into the cultivation media [36].

The confirmation that AmyBS enzyme activity remains unaffected in the presence of both Ca²⁺ and EDTA underscores its independence from calcium. Moreover, AmyBS is capable of breaking down raw starch, albeit at a slightly slower rate compared to soluble starch, as indicated in Table 2. These two characteristics are a significant advantage from an industrial standpoint [43].

Ring et al. [44] demonstrated that starch hydrolysis followed the order of wheat > corn > pea > potato, indicating a correlation with increasing granule size. In contrast, AmyBS exhibits a different order of substrate preference: potato > wheat > corn > rice. In AmyBs case there is a slight different in the order of substrate preference: potato > wheat > rice > corn (Supplementary Table 2). The main hydrolytic products of potato, wheat, and corn starches are glucose and maltose. AmyBS substrate specificity and hydrolytic pattern make it versatile for various industrial applications. Its applicability extends from baking and brewing to the production of sugar syrups. Moreover, it can be applied in starchy waste processing and pharmaceutical industries.

Many bacterial amylases, including α-amylases from Bacillus subtilis S8 [37] and Bacillus subtilis IFO 3108 [11], are known for their ability to hydrolyze raw starch. However, this enzyme is unstable at acidic pH. On the other hand, the α-amylase from B. acidicola, despite being calcium-independent and capable of cleaving raw starch at acidic pH, tends to produce maltose as the primary hydrolytic product of the reaction [45]. The α-amylase derived from the cultural supernatant of Bacillus subtilis S8–18 closely resembles AmyBS’s characteristics (calcium independence, acid pH stability, and the ability to hydrolyze raw starch). However, a significant limitation arises from the unavailability of the sequence for the α-amylase from Bacillus subtilis S8–18, hindering the direct comparison of its sequence with that of AmyBS (Table 2).

Table 2 Comparison of biochemical properties of α-amylases with respect to AmyBS from Bacillus subtilis 3500

For the C-terminal region, purple shading outlines the presence of the C-terminal region, and blank cells indicate that the data for that particular region is unavailable. For effect with Ca2+, blue shading represents thermostabilizing, green activating and orange inhibition effect, green no effect blank cells show unavailable data. ^*ND results are not determined,

Source	Molecular weight, kDa	C-terminal region	Temperature optimum	pH optimum	End products of hydrolyses	Ca²⁺effect	Reference
Bacillus subtilis 3500	69		65 °C	pH 6.0	glucose, maltose		This study
Bacillus subtilis A28	72		70 °C	pH 6.0	ND		[35]
Bacillus subtilis S8–18	57		60 °C	pH 6.0	glucose, maltose		[37]
Bacillus subtilis DR8806	76		70 °C	pH 5.0	maltose, maltotriose		[36]
Bacillus subtilis KCC103	53		65-70 °C	pH 5.0-7.0	maltooligosaccharides		[46]
Bacillus sp. KR8104	68		ND	pH 6.0	glucose, maltose		[38]
Bacillus sp. YX-1	56		40–50 °C	pH 5.0	ND		[47]
Bacillus sp. GRE1	55		65–70 °C	pH 5.5–6.0	maltose		[48]
Bacillus sp. TS-23	65		60 °C	pH 9.0	ND		[49]
Bacillus acidicola	62		60 °C	pH 4.0	maltose		[45]
Geobacillus thermoleovorans	59		80 °C	pH 5.0	maltose, other oligosacharids		[41]

The outcomes from the assessment of metal effects on enzyme activity reveal that copper (Cu²⁺) is the most potent inhibitor among the tested metals, causing more than 90% inhibition. Following this, iron Fe²⁺, Cd²⁺, and Zn²⁺ exhibit substantial inhibitory effects, leading to a reduction in enzyme activity by more than 50%. Notably, Na⁺ is the only metal demonstrating a weak activating effect among the investigated metals (Fig. 5a). An inhibitory effect of Cu²⁺ was also observed in α-amylase of Geobacillus thermoleovorans [41], Bacillus sp. TS-23 [49], Bacillus subtilis A28 [35] and Bacillus sp. GRE1[48].

For potential applications in detergent industries, it is crucial for the enzyme to exhibit stability in various detergents (Fig. 5b). Notably, SDS (1%) exhibited a significant inhibitory effect, among the tested detergents, leading to 80% inhibition of enzyme activity. In contrast, Triton X-100, Triton X-405, and Tween 80 had only a minor impact on the enzyme, suggesting a higher degree of tolerance to these detergents. Similar results have been obtained for Bacillus subtilis A28 α-amylase [35]. In contrast, amylases of Bacillus subtilis DR8806 [36] and Bacillus sp. TS-23 [49] is resistant to SDS.

Thus, the α-amylase gene of Bacillus subtilis strain MDC 3500 was cloned and expressed in Escherichia coli cells. Several structural, phylogenetic, physicochemical, and catalytic characteristics of the enzyme have been studied in detail. It has been shown that the enzyme can be used in many starch-processing processes. The high yield of maltose and glucose is an important characteristic of the new recombinant α-amylase offering promising applications across various industries.

Funding This work was supported by the State Committee of Higher Education and Science of MESCS RA in the frame of the research projects 13-2I359, 16YR-2I016, and 21AA-2I029.

Competing Interests The authors have no relevant financial or non-financial interests to disclose.

Author Contributions Study conceptualization: Ani Paloyan, Tigran Soghomonyan, Lusine Melkonyan and Lev Khoyetsyan, Investigation: Initial microbial strain identification and characterization: Tamara Davidyan and Valeri Bagiyan, Molecular Biology: Ani Paloyan, Protein purification and characterization: Tigran Soghomonyan Anna Mkhitaryan and Lev Khoyetsyan, Resources: Ani Paloyan and Artur Hambardzumyan, Writing – Original Draft: Ani Paloyan, Writing – Review and Editing: Ani Paloyan, Tigran Soghomonyan, Lusine Melkonyan, Lev Khoyetsyan, Anna Mkhitaryan, Artur Hambardzumyan, Tamara Davidyan and Valeri Bagiyan.

Ethical Approval is not applicable.

Consent to participate is not applicable.

Consent to publish is not applicable.

Henrissat B (1991) A classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem J 280, 309–316.
Van Der Maarel MJEC, Van Der Veen B, Uitdehaag JCM, Leemhuis H & Dijkhuizen L (2002) Properties and applications of starch-converting enzymes of the α-amylase family. J Biotechnol 94, 137–155.
Dhanya Gangadharan, Carlos Ricardo Soccol & Ashok Pandey (2006) a-Amylases from Microbial Sources – An Overview on Recent Developments. Food Technol Biotechnol 44 (2), 173–184.
Takata H, Kuriki T, Okada S, Takesada Y, Iizuka M, Minamiura N & Imanaka T (1992) Action of neopullulanase. Neopullulanase catalyzes both hydrolysis and transglycosylation at alpha-(1----4)- and alpha-(1----6)-glucosidic linkages. J Biol Chem 267, 18447–18452.
Gupta R, Gigras P, Mohapatra H, Goswami VK & Chauhan B (2003) Microbial α-amylases: a biotechnological perspective. Process Biochem 38, 1599–1616.
Pandey A, Nigam P, Soccol CR, Soccol VT, Singh D & Mohan R (2000) Advances in microbial amylases. Biotechnol Appl Biochem 31, 135.
Burhan A, Nisa U, Gökhan C, Ömer C, Ashabil A & Osman G (2003) Enzymatic properties of a novel thermostable, thermophilic, alkaline and chelator resistant amylase from an alkaliphilic Bacillus sp. isolate ANT-6. Process Biochem 38, 1397–1403.
Aguilar G, Morlon-Guyot J, Trejo-Aguilar B & Guyot JP (2000) Purification and characterization of an extracellular α-amylase produced by Lactobacillus manihotivorans LMG 18010T, an amylolytic lactic acid bacterium. Enzyme Microb Technol 27, 406–413.
Kunamneni A & Singh S (2005) Response surface optimization of enzymatic hydrolysis of maize starch for higher glucose production. Biochem Eng J 27, 179–190.
Puspasari F, Radjasa OK, Noer AS, Nurachman Z, Syah YM, Van Der Maarel M, Dijkhuizen L, Janeček Š & Natalia D (2013) Raw starch-degrading α-amylase from Bacillus aquimaris MKSC 6.2: isolation and expression of the gene, bioinformatics and biochemical characterization of the recombinant enzyme. J Appl Microbiol 114, 108–120.
Mitsuiki S, Mukae K, Sakai M, Goto M, Hayashida S & Furukawa K (2005) Comparative characterization of raw starch hydrolyzing α-amylases from various Bacillus strains. Enzyme Microb Technol 37, 410–416.
Kaneko A, Sudo S, Takayasu-Sakamoto Y, Tamura G, Ishikawa T & Oba T (1996) Molecular cloning and determination of the nucleotide sequence of a gene encoding an acid-stable α-amylase from Aspergillus kawachii. J Ferment Bioeng 81, 292–298.
Knox AM, Du Preez JC & Kilian SG (2004) Starch fermentation characteristics of Saccharomyces cerevisiae strains transformed with amylase genes from Lipomyces kononenkoae and Saccharomycopsis fibuligera. Enzyme Microb Technol 34, 453–460.
Feller G, Le Bussy O & Gerday C (1998) Expression of Psychrophilic Genes in Mesophilic Hosts: Assessment of the Folding State of a Recombinant α-Amylase. Appl Environ Microbiol 64, 1163–1165.
Chen J, Chen X, Dai J, Xie G, Yan L, Lu L & Chen J (2015) Cloning, enhanced expression and characterization of an α-amylase gene from a wild strain in B. subtilis WB800. Int J Biol Macromol 80, 200–207.
Liu X, Jia W, An Y, Cheng K, Wang M, Yang S & Chen H (2015) Screening, Gene Cloning, and Characterizations of an Acid-Stable α-Amylase. J Microbiol Biotechnol 25, 828–836.
Yao D, Su L, Li N & Wu J (2019) Enhanced extracellular expression of Bacillus stearothermophilus α-amylase in Bacillus subtilis through signal peptide optimization, chaperone overexpression and α-amylase mutant selection. Microb Cell Factories 18, 69.
Engler C, Kandzia R & Marillonnet S (2008) A One Pot, One Step, Precision Cloning Method with High Throughput Capability. PLoS ONE 3, e3647.
Iverson SV, Haddock TL, Beal J & Densmore DM (2016) CIDAR MoClo: Improved MoClo Assembly Standard and New E. coli Part Library Enable Rapid Combinatorial Design for Synthetic and Traditional Biology. ACS Synth Biol 5, 99–103.
Altschul S (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25, 3389–3402.
Corpet F, Gouzy J & Kahn D (1999) Browsing protein families via the ‘Rich Family Description’ format. Bioinformatics 15, 1020–1027.
Gouet P (2003) ESPript/ENDscript: extracting and rendering sequence and 3D information from atomic structures of proteins. Nucleic Acids Res 31, 3320–3323.
Tamura K, Stecher G & Kumar S (2021) MEGA11: Molecular Evolutionary Genetics Analysis Version 11. Mol Biol Evol 38, 3022–3027.
Jones DT, Taylor WR & Thornton JM (1992) The rapid generation of mutation data matrices from protein sequences. Bioinformatics 8, 275–282.
Kelley LA, Mezulis S, Yates CM, Wass MN & Sternberg MJE (2015) The Phyre2 web portal for protein modeling, prediction and analysis. Nat Protoc 10, 845–858.
Sehnal D, Bittrich S, Deshpande M, Svobodová R, Berka K, Bazgier V, Velankar S, Burley SK, Koča J & Rose AS (2021) Mol* Viewer: modern web app for 3D visualization and analysis of large biomolecular structures. Nucleic Acids Res 49, W431–W437.
De Castro E, Sigrist CJA, Gattiker A, Bulliard V, Langendijk-Genevaux PS, Gasteiger E, Bairoch A & Hulo N (2006) ScanProsite: detection of PROSITE signature matches and ProRule-associated functional and structural residues in proteins. Nucleic Acids Res 34, W362–W365.
Laemmli UK (1970) Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4. Nature 227, 680–685.
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72, 248–254.
Miller GL (1959) Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugar. Anal Chem 31, 426–428.
Britton HTS & Robinson RA (1931) CXCVIII.—Universal buffer solutions and the dissociation constant of veronal. J Chem Soc 0, 1456–1462.
Pujadas G & Palau J (2001) Evolution of α-Amylases: Architectural Features and Key Residues in the Stabilization of the (β/α)8 Scaffold. Mol Biol Evol 18, 38–54.
Vihinen M & Mantsiila P (1989) Microbial Amylolytic Enzyme. Crit Rev Biochem Mol Biol 24, 329–418.
Rodriguez Sanoja R, Morlon-Guyot J, Jore J, Pintado J, Juge N & Guyot JP (2000) Comparative Characterization of Complete and Truncated Forms of Lactobacillus amylovorus α-Amylase and Role of the C-Terminal Direct Repeats in Raw-Starch Binding. Appl Environ Microbiol 66, 3350–3356.
Tuzlakoglu Ozturk M, Akbulut N, Issever Ozturk S & Gumusel F (2013) Ligase-Independent Cloning of Amylase Gene from a Local Bacillus subtilis Isolate and Biochemical Characterization of the Purified Enzyme. Appl Biochem Biotechnol 171, 263–278.
Emtenani S, Asoodeh A & Emtenani S (2015) Gene cloning and characterization of a thermostable organic-tolerant α-amylase from Bacillus subtilis DR8806. Int J Biol Macromol 72, 290–298.
Kalpana BJ & Pandian SK (2014) Halotolerant, acid‐alkali stable, chelator resistant and raw starch digesting α‐amylase from a marine bacterium Bacillus subtilis S8–18. J Basic Microbiol 54, 802–811.
Salimi A (2012) Investigations on Possible Roles of C-Terminal Propeptide of a Ca-Independent alpha-Amylase from Bacillus. J Microbiol Biotechnol 22, 1077–1083.
Vihinen M, Peltonen T, Iitiä A, Suominen I & Mäntsälä P (1994) C-terminal truncations of a thermostable Bacillus stearothermophilus α-amylase. Protein Eng Des Sel 7, 1255–1259.
Ohdan K, Kuriki T, Kaneko H, Shimada J, Takada T, Fujimoto Z, Mizuno H & Okada S (1999) Characteristics of Two Forms of α-Amylases and Structural Implication. Appl Environ Microbiol 65, 4652–4658.
Mehta D & Satyanarayana T (2013) Biochemical and molecular characterization of recombinant acidic and thermostable raw-starch hydrolysing α-amylase from an extreme thermophile Geobacillus thermoleovorans. J Mol Catal B Enzym 85–86, 229–238.
Danielsson Thorell H, Beyer NH, Heegaard NHH, Öhman M & Nilsson T (2004) Comparison of native and recombinant chlorite dismutase from Ideonella dechloratans. Eur J Biochem 271, 3539–3546.
Paul JS, Gupta N, Beliya E, Tiwari S & Jadhav SK (2021) Aspects and Recent Trends in Microbial α-Amylase: a Review. Appl Biochem Biotechnol 193, 2649–2698.
Ring S, Gee J, Whittam M, Orford P & Johnson I (1988) Resistant starch: Its chemical form in foodstuffs and effect on digestibility in vitro. Food Chem 28, 97–109.
Sharma A & Satyanarayana T (2012) Cloning and expression of acidstable, high maltose-forming, Ca2+-independent α-amylase from an acidophile Bacillus acidicola and its applicability in starch hydrolysis. Extremophiles 16, 515–522.
Nagarajan DR, Rajagopalan G & Krishnan C (2006) Purification and characterization of a maltooligosaccharide-forming α-amylase from a new Bacillus subtilis KCC103. Appl Microbiol Biotechnol 73, 591–597.
Liu XD & Xu Y (2008) A novel raw starch digesting α-amylase from a newly isolated Bacillus sp. YX-1: Purification and characterization. Bioresour Technol 99, 4315–4320.
Haki GD, Anceno AJ & Rakshit SK (2008) Atypical Ca2+-independent, raw-starch hydrolysing α-amylase from Bacillus sp. GRE1: characterization and gene isolation. World J Microbiol Biotechnol 24, 2517–2524.
Lo H-F, Lin L-L, Chen H-L, Hsu W-H & Chang C-T (2001) Enzymic properties of a SDS-resistant Bacillus sp. TS-23 α-amylase produced by recombinant Escherichia coli. Process Biochem 36, 743–750.

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Molecular Cloning and Biochemical Characterization of a novel thermostable α-amylase of Bacillus subtilis MDC 3500 isolated from acidic soils in Armenia

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Abstract

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Introduction

Materials and methods

Reagents

Bacterial strains and vectors

Cloning and sequence analyses

Protein expression and purification

Characterization of AmyBS

Determination of the Hydrolysis Products

Statistical analysis

Nucleotide sequence accession number

Results

Substrate degradation pattern of AmyBS

Discussion

Conclusion

Declarations

References

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