International Journal of Biological Macromolecules 72 (2015) 290–298

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Gene cloning and characterization of a thermostable organic-tolerant ␣-amylase from Bacillus subtilis DR8806 Shamsi Emtenani a , Ahmad Asoodeh a,b,∗ , Shirin Emtenani a a b

Department of Chemistry, Faculty of Sciences, Ferdowsi University of Mashhad, Vakil-Abad Blv., Mashhad, Razavi Khorasan 9177948974, Iran Institute of Biotechnology, Ferdowsi University of Mashhad, Mashhad, Iran

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Article history: Received 7 June 2014 Received in revised form 18 August 2014 Accepted 19 August 2014 Available online 26 August 2014 Keywords: ␣-Amylase Bacillus subtilis DR8806 Cloning Organic solvent Maltotriose, Thermotolerant

a b s t r a c t The gene encoding an extracellular ␣-amylase from Bacillus subtilis DR8806 was cloned into pET28a(+) vector and expressed in Escherichia coli BL21 (DE3). The recombinant enzyme with molecular mass of 76 kDa exhibited optimal activity at pH 5.0 and 70 ◦ C with high stability in pH and temperature ranges of 4.0–9.0 and 45–75 ◦ C. The enzyme showed a half-life of 125 min at 70 ◦ C. The ␣-amylase activity enhanced in the presence of Na+ , K+ , and Ca2+ ions, while Zn2+ , Pb2+ , and Hg2+ ions inhibited the activity. The recombinant ␣-amylase exhibited high stability towards ioninc detergents sodium dodecyl sulfate (SDS) and cetyl trimethylammonium bromide (CTAB). Organic solvents in reaction media increased the ␣-amylase activity. TLC analysis showed that maltoriose and maltose were the major end products of enzymatic starch hydrolysis. Presenting various properties of recombinant ␣-amylase makes it well suited as a potential candidate for industrial usages. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Carbohydrate-converting enzymes produced by a broad spectrum of living organisms from plants to microorganisms are of significant importance from industrial and biotechnological points of view [1]. Different micoorganisms such as Bacillus species secrete extracellular starch-hydrolyzing enzymes including ␣-amylase, ␤amylase, and ␣-glucosidase to utilize starch as a main energy source [2]. Belonging to the family 13 (GH-13 or GH-H) of glyosyl hydrolases, ␣-amylases catalyze the hydrolysis of ␣-1,4 glycosidic linkages in an endo-acting manner, releasing a mixture of oligosaccharides. ␣-Amylases fit a wide range of applications in starch processing, brewing, baking, and detergent industries by which they rank the first in terms of commercial utilization [3]. ␣-Amylases with desirable features such as thermal stability, substrate specificity, a broad pH profile, improved activity, and proper resistance against denaturing agents and heavy metals extensively draw the attention in industry [4]. Starch processing generally employs thermostable, acidophilic ␣-amylases in liquefaction step. As the activity and stability of most industrial amylases currently utilized in starch processing decline at low pH, starch

∗ Corresponding author at: Department of Chemistry, Faculty of Sciences, Ferdowsi University of Mashhad, Vakil-Abad Blv., Mashhad, Razavi Khorasan 9177948974, Iran. Tel.: +98 51 38795457; fax: +98 51 38795457. E-mail address: [email protected] (A. Asoodeh). http://dx.doi.org/10.1016/j.ijbiomac.2014.08.023 0141-8130/© 2014 Elsevier B.V. All rights reserved.

liquefaction step is restricted to function at pH around 6.50 that is higher than the pH of starch slurry. Thus, the identification and exploitation of microbial thermostable, acidophilic ␣-amylases (active at pH less than 6.50) are of great demand [5,6]. Moreover, hydrolyzing enzymes including proteases and ␣-amylases capable of removing protein/starch-based stains have been dominantly offered on thriving detergent market. Today, various commercial laundry/dishwashing detergents contain cocktails of enzymes that improve washing performance and the environmental compatibility of detergents, indicating the increasing demand for exploiting well-suited hydrolyzing enzymes in detergent formulations [7,8]. Organic solvents and ionic liquids (ILs) have recently garnered widespread attention as attractive solvents in terms of chemical and biotechnological applications. Operating organic solvents as non-aqueous media for enzymatic reactions has numerous advantages such as the improved thermal stability, the inhibition of undesirable water-dependent side reactions, and also the elimination of microbial contamination. However, there are few reports on thermoacidophilic amylases with orgaic solvent tolerance [9,10]. Hence, the isolation of organic solvent-tolerant enzymes seem to be biotechnologically advantageous. On the other hand, ILs, known as environmental friendly green solvents, are now considered as more desirable carbohydrate conversion media compared to conventional organic solvents, particularly in non-aqueos enzymology. Comprehending the activity and stability of starch-hydrolyzing enzymes in ILs may lead to advances in carbohydrate processing [11].

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Overall, the level of production of wild-type enzymes is generally low; therefore, recombinant DNA technology provides the production of recombinant proteins for both basic research and commercial purposes [1]. In the present study, we cloned and expressed an ␣-amylase encoding gene from thermophilic strain Bacillus subtilis DR8806, which was previously isolated from Dig Rostam hot mineral spring in Iran, in mesophilic host, E. coli [12]. Furthermore, biochemical properties of recombinant ␣-amylase were characterized and its stability towards organic solvents, ILs, and commercial detergents as well as its catalytic mode of action on starch were determined. 2. Materials and methods 2.1. Baterial strains and vectors The strain B. subtilis DR8806 (acquisition number: IBRCM10742, Iran) was used as the source of ␣-amylase gene. E. coli DH5␣ and E. coli BL21(DE3) were used as host strains for cloning and expression, respectively. The plasmid pTZ57RT (Fermentas, Maryland, USA) containing an ampicilin-resistance gene was used as cloning/sequencing vector. The vector pET28a(+) (Novagen, USA) bearing kanamycin-resistance gene was exploited for heterogeneous protein expression. B. subtilis DR8806 and E. coli strains were grown aerobically at 37 ◦ C in Lauria–Bertani (LB) medium (1% w/v peptone, 0.5% w/v yeast extract, and 1% w/v NaCl, pH 7.2). 2.2. Molecular gene cloning Chromosomal DNA of B. subtilis DR8806 was extracted as described [13]. A set of two oligonucleotide primers was designed according to multiple alignment of ␣-amylase gene sequences from Bacillus species to isolate ␣-amylase gene. The complete open reading frame (ORF) of ␣-amylase gene (Amy8806) was amplified using forward primer 5 -CGCGGATCCGCGATGTTTGCAAAACGATTCAAAACC-3 and reverse primer 5 CCCAAGCTTGGGTCAATGGGGAAGAGAACCGC-3 containing BamHI and HindIII restriction sites, respectively. Amplification was carried out by Thermocycler (Bio-Rad, CA, USA) under following program: denaturation at 94 ◦ C for 5 min followed by 35 cycles at 94 ◦ C for 45 s, 68 ◦ C for 45 s, and 72 ◦ C for 2 min and a 10 min-final extension at 72 ◦ C. The isolated fragment was then inserted into pTZ57R/T vector for cloning and nucleotide sequencing. Positive colonies of E. coli DH5␣ carrying pTZ-Amy8806 plasmids were first confirmed by colony PCR and double digestion. Afterwards, the target gene was sequenced by automated DNA sequencing. Amy8806 fragment was cloned into pET28a(+) expression vector and transferred into E. coli BL21(DE3). 2.3. Expression and purification of recombinant ˛-amylase E. coli cells carrying recombinant pET28a(+) plasmids were cultivated overnight in LB medium at 37 ◦ C with stirring at 180 rpm. An aliquot of overnight culture (1% v/v) was inoculated into fresh LB medium and incubated at 37 ◦ C to reach OD600 0.5–0.7. To induce the expression, 1 mM isopropyl-␤-d-thiogalactopyranoside (IPTG) was added to growing culture followed by additional 8 h-incubation at 25 ◦ C. The induced cells were collected by centrifugation (12,000 × g, 20 min, 4 ◦ C) and resuspended in buffer A (50 mM NaH2 PO4 , 300 mM NaCl, 10 mM imidazole and 1 mM PMSF, pH 8.0) followed by sonication (6 cycles of intermittent sonication at 25 W for 30 s). The cell lysate was centrifuged at 12,000 × g for 20 min and the supernatant containing soluble recombinant protein was collected. The clear supernatant was loaded onto a

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pre-equilibrated Ni2+ -NTA affinity column (Qiagen, CA, USA). Following the consecutive washing of column with buffer B (50 mM NaH2 PO4 , 300 mM NaCl, and 20 mM imidazole, pH 8.0), target proteins were recovered by elution with buffer C (50 mM NaH2 PO4 , 300 mM NaCl, and 250 mM imidazole, pH 8.0). All purification steps were performed at 4 ◦ C. 2.4. Characterization of recombinant ˛-amylase 2.4.1. Protein quantification and enzyme assay Protein concentration was determined according to the method of Bradford [14] by reading the absorbance at 595 nm with bovine serum albumin as standard. ␣-Amylase activity was assayed in accordance with Bernfeld method [15] using dinitrosalicylic acid (DNSA) reagent. A reaction mixture containing purified enzyme and 1% (w/v) soluble potato starch (Merck, Germany) as substrate in sodium acetate buffer (50 mM, pH 5.0) was incubated at 70 ◦ C for 10 min. The enzyme activity was determined by measuring reducing sugars released following starch hydrolysis at 540 nm. One unit of enzyme activity was defined as the amount of protein needed to liberate 1.0 ␮mol of reducing sugar/min under the assay condition. 2.4.2. Electrophoresis and zymogram analysis The purity and molecular mass of recombinant ␣-amylase were assessed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) as described [16]. Using molecular mass standards (Vivantis, CA, USA), the molecular mass of recombinant protein was estimated on a 12% gel. Amylolytic activity was detected on 10% polyacrylamide gel supplemented with 0.5% starch under non-reducing condition. Non-heated samples were mixed with 6× loading buffer a ratio of 5:1 (v/v). After electrophoresis, a washing solution containing 2.5% Triton X-100 was used to remove SDS from acrylamide gel. Upon staining the gel with Lugol’s solution, purified enzyme was visualized as white area in a dark blue background representing amylolytic activity. 2.4.3. pH and temperature studies The effect of pH on Amy8806 activity was measured at pH range of 2.0–10.0 using different 50 mM buffer systems (glycine–HCl buffer pH 2.0–3.0, sodium acetate buffer pH 3.5–5.5, sodium phosphate buffer pH 6.0–7.5, Tris–HCl buffer pH 8.0–9.5, and Na2 HPO4 –NaOH buffer pH 10.0). ␣-Amylase pH profile was obtained by incubating the enzyme in each buffer system for 10 min at 70 ◦ C with 1% soluble starch as substrate, followed by measuring the amylolytic activity. The enzyme pH stability was studied by pre-incubating the ␣-amylase for 60 min in mentioned buffers and subsequently measuring the activity at 70 ◦ C. The enzyme activity at the beginning of the reaction was taken as 100%. Optimum temperature assays were performed at various temperatures ranging from 30 to 90 ◦ C. The reaction mixture containing the purified enzyme and 1% starch was incubated at different temperatures (pH 5.0). Afterwards, the amount of reducing sugar liberated was determined. For the measurement of thermal stability, the enzyme was pre-incubated at respective temperatures for 60 min. After cooling samples, the residual activity was measured using 1% soluble starch. To evaluate enzyme half-life, ␣-amylase was pre-incubated in sodium acetate buffer (pH 5.0) at varying temperatures (60–80 ◦ C) for 150 min following withdrawn of aliquots at 30 min-intervals. After cooling on ice, enzyme aliquots were assessed for residual activity, as formerly described. 2.4.4. Effect of various metal ions, enzyme inhibitors, and denaturing agents on ˛-amylase activity The influence of different metal cations as well as a number of inhibitors and additives on ␣-amylase activity was studied. An

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aliquot of enzyme solution was pre-incubated with each reagent for 30 min. Subsequently, ␣-amylase activity was measured as described previously. The enzyme activity assayed in the absence of additive was assumed as 100%. 2.4.5. Influence of organic solvents and ILs on ˛-amylase activity The amylolytic activity was investigated in order to study the effect of polar/non-polar organic solvents and imidazolium-based ILs as reaction media for starch hydrolysis. Enzyme aliquots were pre-incubated in different organic solvents (10% and 20% v/v in buffer) and ILs (2–10% v/v in buffer) at ambient temperature for 30 min under 150 rpm stirring. The remaining activity was subsequently calculated according to aforementioned procedure. The activity of control sample (lack of any organic solvents or ILs) was assumed as 100%. 2.4.6. Enzyme performance in the presence of commercial detergents The stability and compatibility of recombinant ␣-amylase with commercial solid and liquid detergents were assayed [7]. To simulate washing conditions, solid detergents including Barf (Paxan, Iran), Softlan (Pakshoo, Iran), Vash (Henkel, Germany), Persil washing powder and handwash powder (Henkel, Germany), Pril (Henkel, Germany), Shoma (TolyPers, Iran), Finish (Reckitt Benckiser, Canada), and Darya (TolyPers, Iran) were dissolved in tap water at final concentration of 5 mg/mL. Additionally, a number of commercial liquid detergents such as Goli (Paxan, Iran), Persil (Henkel, Germany), Ave (Pakshoo, Iran), and Ganj (RaminGostar, Iran) were diluted 100-fold followed by a heat-inactivation of commercial enzymes within detergents at 80 ◦ C for 30 min. An aliquot of purified ␣-amylase was pre-incubated in detergent solutions for 60 min at 50 ◦ C and pH value of 7.5–8.0. Afterwards, the residual activity was determined in the presence of 1% starch and compared with residual activity of Bacillus licheniformis ␣-amylase (BLA). The enzyme activity of control sample (without detergent), incubated under similar conditions, was taken as 100%. 2.4.7. Thin-layer chromatography (TLC) of enzyme end products The end products of starch hydrolysis by recombinant ␣amylase of B. subtilis DR8806 were analyzed using TLC according to the method of Zhang [17]. Reaction mixtures containing 1% soluble starch and pure enzyme were incubated for 3, 6, 18 and 24 h at 70 ◦ C. Defined amounts of reaction mixtures were applied on pre-coated silica gel plate (Merck 60 HPTLC plate, Darmstadt, Germany) in a solvent system composed of n-butanol/acetic acid/water (4:8:1, v/v) at room temperature. Spots were visualized by immersing the air-dried plate in a developing system consisting of 37.5% HCl (1 ml), aniline (2 ml), 85% H3 PO4 (10 ml), ethyl acetate (100 ml), and diphenylamine (2 g), followed by heating at 110 ◦ C in a hot-air oven for 10 min. Glucose (G1), maltose (G2), maltotetraose (G4), and maltohexose (G6) were used as standards. Furthermore, the product specificity of recombinant ␣-amylase in the presence of four imidazolium-based ILs (10% v/v) and some tested commercial detergents was evaluated using TLC. Following the enzyme incubation with the mentioned agents for 30 min, the enzymatic hydrolysis of starch was carried out under assay conditions. Similar to starch hydrolysis assay, a certain volume of reaction mixture was applied on TLC plate and finally the end products of starch hydrolysis released by the treated enzyme were determined and compared to standards. 2.4.8. Substrate specificity Enzyme preference towards different polysaccharides including glycogen (Merck, Germany), ␣-cyclodextrin, ␤-cyclodextrin (Merck, Germany), wheat starch, and corn starch was studied. Substrate (1% w/v) prepared in sodium acetate buffer (50 mM, pH

5.0) was incubated with recombinant enzyme and the measurement of ␣-amylase activity was carried out under assay conditions. A value of 100% activity was considered for soluble starch hydrolysis. 3. Results and discussion 3.1. Construction of plasmid and sequence analysis of ˛-amylase gene ␣-Amylase gene was amplified using genomic DNA of B. subtilis DR8806 as template resulting in an about 2000 bp DNA fragment. The target gene was ligated into pTZ57RT cloning vector and transformed to E. coli DH5␣ for cloning and sequence determination. The Amy8806 sequence was submitted to the GenBank nucleotide sequence database under the accession number KC262177. The deduced primary structure of the protein encoded by Amy88 gene is shown in Fig. 1. Sequence analysis revealed an ORF of 1980 nucleotides encoding a protein composed of 659 amino acid residues. The molecular mass of recombinant protein was predicted to be 72.49 kDa with a theoretical pI of 5.80 using ExPASy server (http://web.expasy.org/protparam/). Detected by SignalP 4.0 software, putative signal peptide was proposed to comprise 33 amino acid residues followed by a cleavage site between positions 33 (Ala) and 34 (Glu). Using CLC Main Workbench program (ver. 6.6.1, Denmark), ␣-amylase amino acid sequence of B. subtilis DR8806 was multiply aligned with other ␣-amylase sequences of various bacteria belonging to genu Bacillus and Geobacillus (Fig. 2a) in which highly conserved regions (regions I–VII), catalytic residues, and functionally essential residues are demonstrated. All members of ␣-amylase superfamily possess three domains consisting of catalytic domain A folded in the form of a (␣/␤)8 -barrel plus domains B and C. Bacillus ␣-amylases show seven conserved segments in the primary structure which mostly includes 138 DAVINH143 (located at strand ␤3 of catalytic barrel), 213 GFRYDAAKH221 (strand ␤4), 245 FQYGEILQ252 (strand ␤5), 305 WVESHD310 (strand ␤7), 185 LYDWNT190 (loop3), 74 GYAAIQTSP82 (strand ␤2), and 340 STPLFFSRP348 (strand ␤8). A catalytic triad comprising acidic residues of Glu (region II), Asp (region III), and Glu (region IV) is compeletly conserved within ␣-amylase superfamily [18,19]. Moreover, two functionally important histidine residues (His143 and His309) being critical for transition-state stabilization are highly conserved. Although the ␣-amylase family can be characterised by several well-conserved sequence regions, only four amino acid residues are totally conserved invariantly throughout GH-13 family which involve the three catalytic residues as well as the arginine (Arg215) in the position i-2 with respect to catalytic nucleophile Asp (region III). Moreover, the phylogenetic tree was constructed by the neighborjoining method using different protein sequences of B. subitilis ␣-amylases (Fig. 2b). 3.2. Affinity purification of recombinant protein and zymogram analysis Recombinant pET28a(+)-Amy8806 vector was transformed to E. coli BL21(DE3) as an expression host. The recombinant ␣-amylase was expressed under the control of IPTG-inducible T7 promoter. ␣-Amylase gene was inserted in frame with an N-terminal region including six histidine residues that allowed the purification of recombinant protein using Ni2+ -NTA affinity chromatogrphy. Single step purification of recombinant enzyme resulted in a specific activity of 3272 U/mg and a yield of 60%. The extracellular ␣amylase activity in the culture broth was negligible, indicating that most part of recombinant protein remained within the host

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Fig. 1. The deduced amino acid sequence of ␣-amylase from Bacillus subtilis DR8806. The well-conserved regions (I–VII) are indicated within colored boxes. Three catalytic residues Asp217 (region II, strand ␤3), Glu249 (region III, strand ␤5), and Asp310 (region IV, strand ␤7) are marked with asterisk. Residues involved in the primary and secondary Ca2+ binding sites are indicated by diamond and triangle, respectively.

cell carrying Amy8806 gene. By comparison, a recombinant ␣amylase bearing an N-terminal His-tag from B. halodurans LBK 34 was purified with a 47% yield and specific activity of 1200 U/mg [20]. Recombinant Amy8806 exhibited a single band with an apparent molecular mass of 76 kDa on 12% SDS-PAGE gel that is slightly larger than the predicted molecular mass (Fig. 3a), since an extra sequence containing 6×His tag added 3.8 kDa to protein mass. Thus, the correct expression of ␣-amylase coding sequence was confirmed. The molecular mass of most amylases are typically ranged in 10–210 kDa [3,21]. ␣-Amylase activity staining of recombinant enzyme indicated a clear zone following enzymatic hydrolysis of starch (Fig. 3b). 3.3. Effect of pH and temperature on ˛-amylase activity and stability The optimum pH of recombinant a ␣-mylase was determined over a pH range of 2.0–10.0. According to pH-activity profile shown in Fig. 4a, maximum activity was observed at pH 5.0. The relative activities at pH 3.0 and 9.0 were 64% and 80%, respectively, of that at pH 5.0, although the enzyme activity rapidly declined at extremely basic pH. According to pH-stability profile depicted in Fig. 4b, DR8806 ␣-amylase was highly stable in pH range of 4.0–9.0 with >80% residual activity. The recombinant ␣-amylase retained 100% of its amylolytic activity between pH 4.0 and 7.0 whereas at pH 3.0 and 9.0, the ␣-amylase retained 77% and 81% of its original activity, respectively. The effect of various temperatures on the ␣-amylase activity (temperature-activity profile) is illustrated in Fig. 4c. The

recombinant Amy8806 showed the highest activity at 70 ◦ C. The enzyme was active at 35–75 ◦ C, while its activity declined at higher temperatures. At 65 ◦ C and 75 ◦ C, relative activities were 92% and 74%, respectively. After 1 h-preincubation at different temperatures, ␣-amylase was found to be quite stable in a wide temperature range from 30 to 85 ◦ C (Fig. 4d); the enzyme retained 100% of its activity when kept at optimum pH and temperature, while retaining >50% of its initial activity at 75–80 ◦ C. A few thermoacidophilic amylases from Bacillus and Geobacillus species have been cloned and characterized including Geobacillus thermoleovorans (pH 5.0 at 80 ◦ C) [19], Bacillus licheniformis NH1 (pH 6.5 at 90 ◦ C) [22], and Bacillus acidicola (pH 4.0 at 60 ◦ C) [21]. Furthermore, according to thermal inactivation experiments, Amy8806 showed a halflife value of 125 min at 70 ◦ C. After 120 min, a rapid decline in enzyme activity (10 min) [6] and Bacillus sp. Ferdowsicous (75 min) [23]. The optimal activity and stability of ␣-amylase at low pH values is of significant importance from industrial point of view. ␣-Amylase with enzymatic performance at acidic pH of natural starch slurry (pH 4.5), as the alternative to currently-used liquefying amylases with optimal pH of 6.8, seem essential to fulfill industrial demands. Additionally, by utilizing acid-stable amylases, the costly and time-consuming step of pH adjustment to 4.5 for saccharification process would be omitted and by-product formation (usually at higher operation pH) would reduce [24]. Consequently, recombinant ␣-amylase with optimal activity in acidic and high tempareture conditions would be a good candidate for enzymatic starch processing in combination with a glucoamylase.

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Fig. 2. (a) The well-conserved regions (I–VII) of ␣-amylase sequences obtained by multiple alignment of amino acid sequences from B. subtilis DR8806 (KC262177), B. subtilis (AFD33644.1), Bacillus KR-8104 (ACD93218.3), B. vallismortis (WP 010331366.1), B. tequilensis (WP 024715525.1), B. amyloliquefaciens (WP 014416850.1), B. methylotrophicus (WP 025650188.1), B. atrophaeus (WP 010788973.1), B. acidicola (JN680873.1), B. licheniformis strain NH1 (EF125542.1), Geobacillus thermoleovorans strain NP54 (JQ409473.1), and Geobacillus thermodenitrificans (ABX83871.1). The catalytic triad (Asp217, Glu249, and Asp310), invariant arginine (Arg215) and conserved histidine residues (His143 and His309) are marked by asterisk, circle, and rectangle, respectively. (b) The phylogenetic tree of ␣-amylase amino acid sequences of Bacillus subtilis strains. Protein sequences of the corresponding ␣-amylases from B. subtilis strains obtained from GenBank were incorporated into the tree by using the neighbor-joining method. Each name at the termini represents the species from which the ␣-amylase protein sequence originated. The accession numbers for each sequence are as follows: B. subtilis DR8806 (KC262177), B. subtilis (WP 003241207.1; 93% homology), B. subtilis (WP 019257405.1; 94%), B. subtilis (WP 029973920.1; 86%), B. subtilis (WP 014112574.1; 94%), and B. subtilis (AFD33644.1; 86%). The sequence of Geobacillus thermodenitrificans (ABX83871.1) was used as the out-group.

3.4. Influence of metal ions and enzyme inhibitors on ˛-amylase activity The effect of numerous metal cations and inhibitors on recombinant ␣-amylase was assessed and the results are summarized in Table 1. Among metal ions tested, ␣-amylase activity enhanced in

Fig. 3. (a) SDS-PAGE analysis of recombinant ␣-amylase expressed in E. coli. M: marker, Lane 1: purified fraction of recombinant protein following affinity purification, Lanes 2–5: cell fractions at 0 (lane 2), 4 (lane 3), 6 (lane 4), 8 (lane 5) hour-post induction with 1 mM IPTG. (b) Starch-zymogram of recombinant enzyme with amylolytic activity.

the presence of 5 mM Na+ , K+ , and Ca2+ ions with remaining activity of 120%, 113%, and 116%, respectively. However, the enzyme activity was strongly inhibited by Zn2+ , Cu2+ , Pb2+ , Hg2+ , and Mn2+ ions and a moderate inhibition was observed by Co2+ and Mg2+ . Thermostable ␣-amylases from Geobacillus thermoleovorans [19] and B. subtilis JS-2004 [25] exhibited similar behavior towards tested metal ions. Calcium ion is generally known to have positive effect on the catalytic activity of most thermostable ␣-amylases and also enhance their themostability, as observed for Amy8806. The inhibition of enzyme activity by copper and mercury ions demonstrated the role of thiol/carboxyl groups as well as the oxidation of indole ring in side chain of aromatic tryptophan within the enzyme, respectively. Worth mentioning, the role of tryptophan and other residues in substrate binding and catalysis has been indicated [19]. Oxidizing agents including potassium iodide and ammonium persulfate exhibited an inhibitory effect on enzyme function (Table 1). Additionally, in the presence of reducing agents such as ascorbic acid and ␤-mercaptoethanol the residual activity was found to be 74% and 59%, respectively. Recombinant ␣-amylase remained 98% of initial activity following incubation with denaturant urea (5 mM). Similar to our enzyme, an ␣-amylase from Geobacillus sp. IIPTN was highly resistant to 5 mM urea [26]. Moreover, a moderate enzyme inhibition was observed by 10% Triton-X100. Interestingly, Amy8806 almost retained its catalytic efficiency in the presence of cationic surfactant CTAB and anionic

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Fig. 4. Effect of pH and temperature on the activity and stability of recombinant ␣-amylase. Activity (a) and stability (b) as a function of pH; the purified enzyme exhibited maximum activity at pH 5.0, retaining 100% of its initial activity after 1 hour-incubation under optimal conditions. Temperature-activity (c) and stability (d) profiles of ␣-amylase at pH 5.0. Maximum amylase activity and stability was observed at 70 ◦ C. Experiments were carried out in triplicate.

surfactant SDS (10% w/v). The enzyme stability towards SDS is of great significance, particularly in detergent formulation, because SDS-resistant amylases have been rarely reported [7]. The enzyme retained 80% of its initial activity in the presence of 5 mM chelating agent EDTA, suggesting that metal ions including calcium are essential for enzyme activity. The inhibitory effect of EDTA has been reported for ␣-amylase from Geobacillus sp. LH8 [27]. It is proposed that through sulfonating serine residues at the enzyme catalytic site, phenylmethylsulphonyl fluoride (PMSF) inhibits the enzymatic activity, as observed for our recombinant ␣-amylase and B. acidicola ␣-amylase [21]. 3.5. Effect of organic solvents and ILs on amylase activity As shown in Table 2, organic solvents as hydrolysis media were found to have a promoting effect on ␣-amylase activity compared to control. The enzyme activity was stimulated by 105%, 144%, and 163% in the presence of 20% (v/v) methanol, ethanol, and butanol, respectively. Moreover, ␣ -amylase activity slightly enhanced by following water miscible solvents: hexane (112%), heptane (104%), and toluene (139%). Similar results have been reported for an amylase from B. agaradhaerens Mi-10-62 which considerably retained its activity in 30% (v/v) organic solvents such as dodecane, heptane, methanol, and propanol [28]. Furthermore, a halophilic ␣-amylase from Nesterenkonia sp. strain F exhibited a proper stability towards 20% (v/v) chloroform and toluene [10]. The hydrophobicity of an organic solvent is detrmined by a parameter termed “log Po/w ” value. Typically, organic solvents having low log Po/w values (watermiscible or polar solvents) exhibit more biological toxicity resulting in more inhibitory effect on biocatalysts compared to high Po/w

solvents (water-immiscible or nonpolar solvents) [29]. In this investigation, no clear relation was obtained between the log Po/w value of tested organic solvents and the stability of recombinant ␣-amylase. Additionally, organic solvents with low log Po/w values (−0.208 to 0.8) revealed a slightly destabilizing effect on Amy8806 activity, whereas nonpolar organic solvents with high Po/w values (>1.9) stimulated ␣-amylase activity. In spite of the advanteges of organic solvents, hydrocarbon-containing environments result in denaturation of most enzymes and loss of their catalytic activity. Consequently, enzymes being intrinsically stable and active in organic solvent solutions appear to be quite attractive for industrial applications such as treatment of carbohydrate-polluted industrial wastewater contaminated with organic solvents [9]. Considering the remained catalytic activity of recombinant amylase, organic solvent-based solutions could be employed as suitable reaction media for carbohydrate hydrolysis. Due to the impact of ILs as dispersion/modification media on enzymatic transformation, the activity of recombinant enzyme was measured towards four imidazolium-based ILs including 1-ethyl-3-methylimidazolium bromide ([EMIm][Br]), 1-butyl-3-methylimidazolium bromide ([BMIm][Br]), 1-hexyl3-methylimidazolium bromide ([HMIm][Br]), and 1-butyl-3methylimidazolium chloride ([BMIm][Cl]) (Fig. 4). The results demonstrated that as the IL concentration increased, the enzyme activity decreased. At 10% (v/v) concentration of ILs, the highest retaining activity (74.7%) was observed in reaction mixture containing [BMIm][Cl], while [HMIm][Br] was found to cause the most inhibitory effect amongst Br-containing ILs (60% retaining activity). Generally, as the chain length of alkyl substituents on imidazolium ring of cation and anion size decrease, the polarity as well as enzyme

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Table 1 Effect of various metal ions and chemical reagents on Amy8808 activity.a Reagent

Relative enzyme activity (%)

Metal ions Hg2+ Ba2+ Co2+ Cu2+ Mg2+ Pb2+ Mn2+ Ca2+ Fe2+ Zn2+ Na+ K+ Oxidizing agents Potassium iodide Ammonium persulfate Reducing agents Ascorbic acid ␤-mercaptoethanol Chelating agents EDTA Sodium citrate Detergents SDS Triton X-100 CTAB Additives Glycerol PEG 4000 Urea Inhibitors DTNB PMSF Dithiothreitol Dimethylfomamide Phenanthroline Tetrafluoroethylene

1 mM 36 81 73 100 44 0 23 120 29 102 115 100 1 mM 39 13 1 mM 67 61 1 mM 85 65 10% 85 45 113 1% 55 71 100 1 mM 71 33 50 42 72 76

5 mM 14 112 38 0 22 0 0 116 26 0 120 113 5 mM 23 0 5 mM 74 59 5 mM 80 32 20% 70 12 93 2% 48 73 98 5 mM 46 23 39 0 64 68

Fig. 5. Amylase activity in the presence of four imidazolium-based ILs (2–10% v/v). Values presented are the means of triplicate analyses.

a Enzyme preparation was pre-incubated with different concentrations of chemical agents and the residual activity was determined under assay conditions. Amylase activity of control sample in the absence of any additives was taken as 100%. The standard errors were less than 5% of the mean.

activity increase [11]. It was previously reported that thermophilic ␣-amylases exhibited more stability and activity in ILs-containing solutions compared to mesophilic amylases [30]. This finding allows [BMIm][Cl] to be used as a dispersion/modification meduim for biocatalysis in enzymatic applications (Fig. 5.). 3.6. Influence of commercial detergents on recombinant ˛-amylase activity In this study, the effect of commercial laundry/dishwashing detergents on the activity of Amy8806 was assessed and compared with B. licheniformis ␣-amylase (BLA). As shown in Fig. 6, Table 2 Effect of various organic solvents (10% and 20% v/v) on amylase activity. Organic solvents

Acetone Methanol Ethanol Butanol Isopropanol Hexane Heptane Isoamylalcohol Diethyl ether Toluene

Relative amylase activity (%) 10% v/v

20% v/v

116 113 128 68 77 112 104 117 99 139

85 105 144 163 105 114 129 64 77 98

Fig. 6. The effect of commercial solid and liquid detergents on the activity of ␣amylase from B. subtilis DR8806 compared with B. licheniformis amylase. The enzyme was first treated with detergent solution and the residual activity was then measured in the presence of substrate. The activity of control samples in the absence of detergents was taken as 100%. All determinations were performed in triplicate.

our recombinant ␣-amylase was totally stable towards both solid and liquid detergents, retaining 82–100% of its initial activity. Interestingly, Amy8806 exhibited good compatibility and activity in the presence of detergents compared with BLA. The residual activity of Amy8806 was 99%, 99%, and 97% following 1 h-preincubation with Ave (Pakshoo, Iran), Persil (Henkel, Germany), and Shoma (TolyPers, Iran) detergents, respectively, while BLA retained 96%, 71%, and 60% of its original activity towards aforementioned detergents under the same conditions. Totally, both amylases exhibited the highest stability towards Ave detergent whereas the minmal stability for Amy8806 and BLA was observed following the enzyme incubation with Persil washing powder and Ganj. By comparison, thermostable ␣-amylase from B. licheniformis NH1 showed good compatibility with various solid/liquid laundry detergents under similar assay conditions [7]. Enzymes retaining active in the presence of potentially inhibitory ingredients, routinely formulated into commercial detergents, are more favored for detergent industry [8]. According to the enzyme stability towards commercial detergents and ionic surfactants like SDS and CTAB as well as its activity up to pH 9.0, recombinant ␣-amylase could be potentially applied in the formulation of liquid and powder laundry/dishwashing detergents to remove starch-containing stains.

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maltooligosaccharides, particlurly maltotriose. In the case of ILs, maltoteriose was the major end product of starch hydrolysis by the treated enzyme (Fig. 7b). Interestingly, as shown in the chromatogram the highest maltoteriose production was observed for sample containing B[MIm]Cl ionic liquid in which the enzyme showed the highest stability. Moreover, recombinant Amy8806 was stable toward the tested detergents and could catalyze the hydrolysis of starch into short lenght maltooligosaccharids during the incubation time (Fig. 7c). 3.8. Substrate specificity analysis Recombinant ␣-amylase from B. subtilis DR8806 was investigated for its ability to hydrolyze different carbohydrates. Among the substrates tested, soluble potato starch was most efficiently hydrolyzed by Amy8806. The recombinant enzyme hydrolyzed some substrates (1% w/v) including soluble potato starch, glycogen, wheat starch, and corn starch with degradation rates of 100%, 65%, 62%, and 43% without any noticeable activity on ˛and ˇ-cyclodextrins. Thus, Amy8806 can hydrolyze ␣-glycosidic bonds on both linear and branched carbohydrate polymers. Similar hydrolyzing tendency was also obtained by Sharma et al. [21], where B. acidicola ␣-amylase hydrolyzed soluble potato starch (100%) and corn starch (68%) without any affinity towards cyclodextrines. To conclude, recombinant ␣-amylase (Amy8806) is a thermostable, acidophilic, and organic solvent-resistant enzyme with high compatibility towards detergents, which suggests its potential applications in different biotechnological fields such as starch processing and detergent formulation. Acknowledgment Authors gratefully appreciate the Institute of Biotechnology and research council of Ferdowsi University of Mashhad - Iran for their financial support (grant numbers: 3/27142; 23-2-1392 and 4065; 06-02-1389).

Fig. 7. (a) TLC analysis of hydrolysis products of starch by B. subtilis DR8806 recombinant ␣-amylase after 3, 6, 18, and 24 h tretaments. Lane 1 (G): standard oligosaccharides (G1, glucose; G2 maltose; G4, maltotetraose; and G6, maltohexose). (b) The product specificity of DR8806 ␣-amylase in the presence of four imidazolium-based ILs (10% v/v). (c) The product specificity of recombinant ␣amylase treated with commercial solid detergents including Barf, Pril, Persil washing powder, and Finish as well as liquid detergents including Ganj and Ave.

3.7. End product profile of starch hydrolysis The end products of starch hydrolysis by Amy8806 were assessed on TLC chromatogram for 3-24 h (Fig. 7a). TLC analysis indicated a range of short length maltooligosaccharides from random degradation of potato starch, gradually converted to maltose (after 24 h treatment) in which maltose (G2) and maltotriose (G3) are the major end products. Based on TLC analysis of the end-products, Amy8806 could be classified as an endo-acting maltose/maltoteriose-forming ␣-amylase, making it suitable to be utilized for the production of high maltose-content maltodextrins from starch. In addition to enzyme staibility assay, the product specificity of Amy8806 was determined in the reaction solutions containing imidazolium-based ILs and a number of commercial detergents. Totally, the results show that the recombinant ␣-amylase not only was stable stable in the presence of tested ILs and commercial detergents, but also could hydrolyze starch into different

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