Biologia 69/4: 419—427, 2014Section Cellular and Molecular BiologyDOI: 10.2478/s11756-014-0343-2

Isolation and production of thermostable α-amylasefrom thermophilic Anoxybacillus sp. KP1 from Diyadin hot springin Agri, Turkey

Fatma Matpan Bekler* & Kemal Guven

Department of Biology, Faculty of Science, Dicle University, 21280 – Diyarbakir, Turkey; e-mail: fatmatpan@hotmail.com

Abstract: A novel amylolytic enzyme producing thermophilic bacterial strain KP1 from the Diyadin hot spring water inAgri, Turkey, was isolated in the present study. Phylogenetic analysis based on the partial 16S rRNA gene, biochemicaland physiological tests revealed that the strain KP1 belongs to the genus Anoxybacillus. The pH and temperature optimafor the α-amylase production by Anoxybacillus sp. KP1 were 8.0 and 50◦C, respectively, where the maximum growth wasobtained at the 28th hour of incubation and the highest α-amylase activity was obtained at the 40th hour of incubation(8979.6 U/mL). The optimum pH and temperature for the enzyme activity were 8.0 and 60◦C, respectively. The maximumα-amylase production was secreted in the presence of 2% (w/v) soluble starch (10837.7 U/mL). Among the various organicand inorganic nitrogen sources tested, while keeping the beef extract concentration constant, casamino acid (14310.6 U/mL),urea (14126 U/mL), and tryptone (13217.2 U/mL) at a concentration of 2% gave the maximum α-amylase production. Theenzyme activity was enhanced in the presence of 1.5 mM Mn2+ (123%), whereas it was strongly inhibited 1.5 mM by Hg2+.Inhibition by 89% was obtained also with sodium dodecyl sulphate (1%). The enzyme was found to be relatively stable ata range of pH and temperature.

Key words: Anoxybacillus; thermophilic bacterium; α-amylase; hot water spring.

Abbreviations: BMA, BMB and BMC, basal medium A, B and C, respectively; SD, standard deviation; SDS, sodiumdodecyl sulphate.

Introduction

Today, because of decreasing natural sources, microor-ganisms are seen as having potential in a lot of pro-duction areas and dense studies are being conductedon this issue. The advantages of enzymes of microbialorigin, because of being economical in industrial pro-duction and not being bound to seasonal and potentiallimitations, have long been defended (Topal et al. 2000).Extracellular enzymes that degrade starch in dif-

ferent environmental niches are produced by a greatnumber of bacteria, fungi, and yeasts. A variety ofpolysaccharide hydrolyzing enzymes suitable for vari-ous industrial applications have appeared in the lastfew decades, thus leading to the screening of these en-zymes for novel properties (Sharma & Satyanarayana2012). Thermostable enzymes, mainly isolated fromthermophilic organisms, have found a lot of commercialapplication fields because of their general intra-stability(Haki & Rakshit 2003). The advantages of such en-zymes are as follows: having high catalytic activities,not producing unwanted side-products, being more sta-ble and cheaper, availability in high amounts and purity(Nascimento & Martins 2004). Moreover, lots of enzy-

matic reactions that are used commonly in industry,are realized at high temperatures. Industrial applica-tions of thermostable amylases have advantages, suchas decreasing contamination risk, increasing diffusionrates, being resistant to denaturing agents, solutions,and proteolytic enzymes (Saxena et al. 2007).

α-Amylase (EC 3.2.1.1) is an enzyme that hydroly-ses internal α-1,4-glycoside linkages of α-glucans, suchas starch and glycogen (Kumagai et al. 2013). Amy-lases are used in filling of pores of texture and papers,desizing of starch in woven texture in textile, in re-moving food and starch stains in dry cleaning, in foodbiotechnology in the following applications: bakery, liq-uefying doughs, production of starch hydrolysis prod-ucts such as glucose and fructose, production of cornand chocolate syrup, production of low-calories beer, inpurification of apple and pear juice, malt production,in removing stickiness in paper industry, in detergentindustry as well as in medical industry (Gupta et al.2003; Ul-Haq et al. 2003; Apar & Ozbek 2004).Currently, scientists have great interest in study-

ing thermophilic Anoxybacillus, since knowledge accu-mulated from fundamental and applied studies suggeststhat this genus can serve as a good alternative in many

* Corresponding author

c©2014 Institute of Molecular Biology, Slovak Academy of Sciences

420 F. Matpan Bekler & K. Guven

environmental, enzyme and biotechnology applications(Goh et al. 2013).All identified Anoxybacillus species are thermo-

philic, Gram-positive, aerobic, spore-forming, rod-shaped bacteria identified differently from Bacillus.To date, identified and amylolytic enzyme produc-ing Anoxybacillus species, which have been depositedin the GenBank database (Benson et al. 2013) areAnoxbacillus rupiensis TSSC-4, Anoxybacillus bep-puensis TSSC-1, Anoxybacillus amylolyticus TSSC-5, Anoxybacillus sp. DT3-1, Anoxybacillus sp. SK3-4, Anoxybacillus pushinoensis (DSM 12423T), Anoxy-bacillus kamchatkensis subsp. asaccharedens subsp.,Anoxybacillus eryuanensis, Anoxybacillus flavithermusyunanensis, Anoxybacillus flavithermus (DSM 2641T),Anoxybacillus ayderensis (NCIMB 13972T), Anoxy-bacillus contaminans (DSM 15866T), Anoxybacillusgonensis (NCIMB 13933T), Anoxybacillus mongolien-sis (DSM 19169T), Anoxybacillus salavatliensis (DSM22626T), Anoxybacillus tengchogensis (KCTC 13721T)and Anoxybacillus kestanbolensis (NCIMB 13971T).The purpose of this study was to isolate ther-

mophilic bacteria from mud and water of Diyadin hotspring (Kopru hot water) in Agri, in North-EasternTurkey. Thermophilic bacteria are well known to beof great importance, since they are the source of in-dustrially used enzymes. Therefore we investigate theindustrially important amylolytic enzyme productionability of these bacteria and certain characteristics ofthis enzyme. In addition, this study attaches great im-portance as Kopru hot water was a source not studiedbefore with regard to bacteria isolation; this will thus bea guiding study in determining the diversity of bacteriaof thermophilic characteristics from geothermal watersof Turkey.

Material and methods

Bacterial strainIn this study, the strain KP1 was used, which was iso-lated from the Kopru hot water (39◦29′35′′ N, 43◦39′1′′ E)(temperature 50◦C, pH 7.4) of Diyadin township of AgriProvince, in North-Eastern Turkey. Thermal mud sampleswere diluted with 1% sterilized water and were then platedonto nutrient broth (Sigma) agar plates. Before the inocu-lation, all of the samples were treated at 80◦C for 10 min inorder to isolate only the spore-forming microorganisms. Theinoculated plates were incubated for 48 h at 55◦C in orderto obtain colonial growth. The cells grown were collectedwith sterile medium and used for the inoculation of 33 mLof medium in 250 mL Erlenmeyer flasks. The selected organ-isms were identified by morphological and some biochemicaland physiological tests, as well as by partial 16S rRNA se-quence. Partial 16S rRNA analyses of the isolates obtainedwere conducted by Ref-Gen (METU Technocity/Ankara).The sequences consisting of about 826 nucleotides of the 16SrRNA gene were determined. These sequences were com-pared with those available in the GenBank database using aBLAST search (http://www.ncbi.nlm.nih.gov/blast/). The16S rRNA gene sequences of the species most closely relatedto our strain were retrieved from the database. Phyloge-netic tree was constructed using the software package CLC

Sequence Viewer 6.0 (http://www.clcbio.com/products/clc-sequence-viewer/).

Growth medium and optimization of the cultureThe inoculum was routinely grown in nutrient brothmedium composed of (g/L): beef extract, 10; peptone, 10;NaCl, 5; the pH was adjusted to 7.0 prior to sterilization.The media were autoclaved at 120◦C for 20 min. The cul-tivations were performed in a shaker for 4–64 h at 55◦C in250 mL Erlenmeyer flasks with a working volume of 33 mL.Growth was determined by measuring the increase in OD470.The effect of pH on the growth was determined in the rangeof 4.0–11.0, with 1.0 pH steps using citric acid (for pH 4.0–6.0), Tris-HCl (for pH 6.0–9.0), and glycine-NaOH (for pH10.0–11.0) buffers at a concentration of 0.1 M. The pH val-ues were adjusted at room temperature. The influence oftemperature on the growth was determined at an optimumpH at different temperatures (30–75◦C) with 5◦C steps un-der shaking.

Amylolytic activityThe cultures were centrifuged at 8,200×g for 10 min at 4◦Cand the cell-free supernatants were used for the estimationof amylolytic enzyme activities. The enzyme activity wasdetermined according to Bernfeld (1955). According to thismethod, 100 µL of enzyme solution and 200 µL of starchsolution at 0.5% (from potato) were incubated for 30 minat 70◦C. At the end of this duration, in order to stop thereaction, 400 µL of 3,5 dinitrosalicylic acid was added andit was kept in a boiling water bath for 5 min. Next, aftermaking a dilution using 3 mL of distilled water, the spec-trophotometric measurement was conducted at 489nm. Oneunit of the amylase activity was defined as the amount ofenzyme that released 1 µmol of maltose per minute per mLunder the assay conditions. The protein content in the ex-tracellular extracts was determined by the Lowry method(1951) using bovine serum albumin as a standard in thisprocedure.

Process optimization for the maximum α-amylase produc-tionIn order to determine the effect of different media on theα-amylase production, 500 µL of the obtained isolate wasinoculated into 25 mL of nutrient broth (25 g/L), basalmedium A (BMA) (0.2% beef extract, 0.2% peptone and0.1% NaCI), basal medium B (BMB) (0.1% beef extract,0.1% peptone and 1% NaCI), and basal medium C (BMC)(2% soluble starch, 0.2% yeast extract, 1% beef extract,0.02% CaCl2 and 0.01% MgSO4.7H2O) in 100 mL Erlen-meyer flasks and cultivated at an optimum temperature ina shaker for 24 h. The α-amylase enzyme activity of thisisolate was then carried out at optimum conditions.

Effect of the incubation time on bacterial growth and α-amylase productionOne hundred mL of BMA was chosen for determining theeffect of incubation time on bacterial growth and α-amylaseproduction at optimum conditions. One mL of the isolatewas inoculated and samples were taken at the end of ev-ery 4th hour over a 48-h period and growth was determinedby measuring the increase in OD470 after which was cen-trifuged at 8,200×g. Bacteria were then precipitated and asupernatant was used for the α-amylase activity.

Thermostable α-amylase from Anoxybacillus sp. KP1 421

Table 1. Some characters that distinguish Anoxybacillus sp. KP1 from other Anoxybacillus species.a

Characteristics KP1 1 2 3 4 5

Gram painting + + + + + +Spore forming + + + + + +Relation to O2 Aerobe Aerobe/Facultative aerobe Aerobe/Facultative aerobe Aerobe Facultative aerobe AnaerobeCell form Rod shape Rod shape Rod shape ND ND Rod shapePigmentation Yellow Yellow ND ND ND NDTemperature range (◦C) 40–65 30–72 45–65 35–60 40–70 37–66Optimum temperature (◦C) 50 60–65 61 50 55–60 62pH range 6.5–9.5 5.5–9.0 5.0–6.5 6.0–11.0 6.0–10.0 8.0–10.5Optimum pH 8.0 7.0 5.6 7.5–8.5 7.5–8.0 9.5–9.7Motile + + + + + –Oxidase + + + + + –Caseine + ND + ND ND –Starch + + + + + +Lipase + ND ND ND ND NDUrease + – ND ND NDGelatine + – – + + –Indole – – ND ND ND NDCatalase + + ND + + –β-Galactosidase + + ND ND ND NDPhosphatase + ND ND ND ND ND

a “+” and “–“ denote positive result or growth and negative result or no growth, respectively, ND, none defined. 1, Anoxybacillusfavithermus DSM 2641 (NR 026516) – Pikuta et al. (2000); 2, Anoxybacillus amylolyticus sp. – Poli et al. (2006); 3, Anoxybacillusayderensis NCIMB 13972T – Dulger et al. (2004); 4, Anoxybacillus gonensis NCIMB 13933T – Belduz et al. (2003); 5, Anoxybacilluspushchinoensis DSM 12423T – Pikuta et al. (2000).

Effect of the carbon and nitrogen sources on the α-amylaseproductionIn order to determine the effect of various carbon and ni-trogen sources on the enzyme production, 500 µL of theobtained isolate was inoculated into 25 mL of BMA con-taining various 2% carbon sources (such as glucose, lactose,sucrose, fructose, galactose and soluble starch) and variousnitrogen sources (such as bacto liver, casamino acid, glycine,tryptone, urea and ammonium sulphate) in 100 mL Erlen-meyer flasks. The α-amylase enzyme activity of this isolatewas carried out.

Effect of various starches and raw starches on the α-amylaseproductionStrain KP1 was grown in BMA liquid medium containingvarious concentrations (0.5%, 1%, 1.5%, 2% and 3%) of dif-ferent starch sources (soluble, potato, rice and wheat starch)and 2% raw starch sources (wheat, corn and rice starch) inorder to study their effect on the enzyme production.

Characterisation of the partially purified enzymeThe amylolytic enzyme was partially purified by 80% ammo-nium sulphate saturation. The characterization of enzymein the following experiments was carried out on partiallypurified enzyme.

Effect of pH on the activity and stability of the α-amylaseThe optimum temperature and pH were measured by acolorimetric method, using soluble starch as a substrate.The effect of pH on the activity of the partially purified α-amylase was measured in the range of 4.0–11.0, using theappropriate buffers at a concentration of 0.1 M (4.0–6.0,sodium citrate; 7.0–8.0, Tris–HCl; and 9.0–10.0, glycine-NaOH) under standard assay conditions. For the measure-ment of the pH stability, enzyme was pre-incubated inbuffers at different pH values in the range of 4.0–11.0 for1 h at 55◦C. Aliquots were withdrawn and the residual amy-lolytic activity was determined under standard assay condi-tions.

Effect of temperature on the activity and stability of the α-amylaseThe effects of temperature on the α-amylase activity wasstudied at temperatures from 20 to 80◦C, using solublestarch as a substrate for 30 min in 0.1 M Tris-HCl bufferat an optimum pH. The thermostability of the partially pu-rified enzyme was examined by incubating the enzyme for30 min at different temperatures. Aliquots were withdrawnat the desired time intervals to test the remaining activ-ity under standard conditions. The unheated enzyme wasconsidered as the control (100%).

Effects of some metal ions and SDS on the α-amylase ac-tivityThe effect of some metal ions and sodium dodecyl sulphate(SDS) on the partially purified (after 80% ammonium sul-phate saturation) amylolytic enzyme activity was studiedby pre-incubating the enzyme in the presence of substanceswith a final concentration of 1.5 mM (Ca2+, Mn2+, Cu2+,Hg2+ and SDS) for 30 min at 37◦C, and then performingthe assay at the optimum temperature. All of the metalsused were in the chloride form. The activity in the absenceof any additives was taken to be 100%. The residual activitywas measured using the standard assay conditions.

Statistical analysisThe results were presented as the mean ± standard devia-tions (SD) of at least 3 experiments. Mann-Whitney U testwas done with the MedCalc Statistical software (for Win-dows; http://www.medcalc.org/).

Results and discussion

Bacterial strainIn recent years, researchers have conducted many stud-ies on isolation of microorganisms that possess ther-mophilic characteristics. With regard to Anoxybacil-lus, initially Pikuta et al. (2000) reported thermophilicAnoxybacillus species as Anoxybacillus favithermus

422 F. Matpan Bekler & K. Guven

Fig. 1. Partial 16S rDNA sequence-based phylogenetic neighbour-joining tree showing the phylogenetic relationship of strain KP1relative to other strains of the genus Anoxybacillus. Bootstrap values (%) from 1,000 replicates are shown. The tree topology wasobtained by calculation using the CLC Sequence Viewer 6 program. Bacillus subtilis is used as the out-group. Bar indicates 0.05nucleotide substitutions per position.

DSM 2641 and Anoxybacillus pushchinoensis DSM12423T and stated that these were different from Bacil-lus species but belonged to Bacillacea. In later studies,Belduz et al. (2003) and Dulger et al. (2004) identifiedthermophilic Anoxybacillus gonensis NCIMB 13933T

and Anoxybacillus ayderensis (NCIMB 13972T) fromgeothermal hot spring water, respectively. Poli et al.(2006) isolated thermophilicAnoxybacillus amylolyticusfrom Rittmann Mountain. Derekova et al. (2007) deter-mined that a new thermophilic Anoxybacillus rupien-sis was isolated from the Rupi Basin hot water spring.Furthermore, Gul-Guven et al. (2008) isolated Anoxy-bacillus kamchatkensis subsp. asaccharedens from hotspring water in Batman. Chai et al. (2012) also isolatedthermophilic Anoxybacillus sp. DT3-1 and SK3-4 fromtwo hot springs in Malaysia. Anoxybacillus beppuensisTSSC-1 was isolated from a clay-type soil sample fromthe hot spring reservoir in Tulsi Shyam, Gujarat, India(Kikani & Singh 2012). They described that the speciesbelong to Anoxybacillus genus and were generally aer-obe (or facultative aerobe), gram-positive, motile, sporeforming, rod-shaped, and positive for catalase, oxidasestarch hydrolysis activity. Table 1 shows some char-acteristics that distinguish the current Anoxybacillussp. KP1 from other Anoxybacillus species. The rela-tionship of partial 16S rRNA sequences of Anoxybacil-lus sp KP1 with other Anoxybacillus species that pro-duce amylolytic enzymes is shown in Figure 1. It canbe seen that the partial 16S rRNA sequences deter-mined for the strain KP1 was identified as Anoxybacil-lus sp. KP1 (GenBank Accession No.: KC525949) iso-lated from Kopru hot water, Diyadin.

Growth optimization of the culture and determinationof amylolytic activityFour different media namely, nutrient broth and BMA,BMB and BMC were used for determining optimalbacterial growth and enzyme production. The high-est α-amylase activity was obtained at 40th h incuba-tion (8979.6 U/mL) in BMA using the Bernfeld mehod(1955). The BMA medium was chosen for following ex-periments. The results of the time-course studies on thegrowth of Anoxybacillus sp. KP1 and the α-amylaseproduction is shown in Figure 2. The maximum growth

Fig. 2. Effect of time-course on bacterial growth and α-amylaseproduction.

Fig. 3. Effect of temperature on bacterial growth.

was obtained at 28th h of incubation for Anoxybacil-lus sp. KP1. It was determined that the optimum tem-perature and pH for the growth of Anoxybacillus sp.KP1was 50◦C (Fig. 3) and 8.0 (Fig. 4), respectively.The similar optimum temperature value was obtained

Thermostable α-amylase from Anoxybacillus sp. KP1 423

Fig. 4. Effect of pH on bacterial growth.

forAnoxybacillus ayderensis NCIMB 13972T (Dulger etal. 2004) and Anoxybacillus contaminans DSM 15866T(De Clerck et al. 2004) as 50◦C, while it was foundto be 54◦C for Anoxybacillus voinovskiensis NCIMB13956T (Yumoto et al. 2004), 55–60◦C for Anoxybacil-lus gonensis NCIMB 13933T (Belduz et al. 2003), 61◦Cfor Anoxybacillus amylolyticus DSM 15939T (Poli et al.2006), 62◦C for Anoxybacillus pushchiensis KT (Pikutaet al. 2000) and finally 65◦C forAnoxybacillus bogroven-sis (Atanassova et al. 2008). The optimum pH forthese species were as follows: 7.5–8.5 for A. ayderen-sis NCIMB 13972T (Dulger et al. 2004) and A. go-nensis NCIMB 13933T (Belduz et al. 2003), 7.0–8.0 forA. voinovskiensis NCIMB 13956T (Yumoto et al. 2004)and 8.0 for A. bogrovensis (Atanassova et al. 2008). Ourfindings demonstrate that the studied strain KP1 is al-kaliphilic and moderately thermophilic Anoxybacillussp.

Effect of the carbon and nitrogen sources on the α-amylase productionAs shown in Figure 5, the best substrate for the α-amylase production for Anoxybacillus sp. KP1 was 2%soluble starch (10745.2 U/mL). Kikani & Singh (2012)investigated the effect of carbon sources on the produc-tion of α-amylase by Anoxybacillus beppuensis TSSC-1and they determined that the production of α-amylasewas increased in the presence of soluble starch. Yanget al. (2012) reported an amylase production increasein the presence of soluble starch for Anoxybacillus sp.YIM 342. Behal et al. (2006) reported that the maxi-mum α-amylase production was increased by fructose.Our findings are supported by the results of previousresearchers. In our study, with glucose, galactose, orsucrose as the carbon source, only a low basal level ofα-amylase was produced and the α-amylase levels de-creased immediately after the addition of the glucose(Fig. 5). It is well known that the synthesis of car-bohydrate degrading enzymes in most species of thegenus Bacillus is subjected to catabolic repression byglucose (Wind et al. 1994; Asgher et al. 2007) and su-crose (Qader et al. 2006).Among the various organic and inorganic nitrogen

sources tested, while keeping the beef extract concen-tration constant, casamino acid (14310.6 U/mL), urea(14126 U/mL) and tryptone (13217.2 U/mL) at a con-centration of 2% gave the maximum α-amylase produc-tion (Fig. 6) for Anoxybacillus sp. KP1. Malhotra et al.(2000), Parakash et al. (2009) and Yang et al. (2012)determined that the production of α-amylase was alsoincreased in the presence of tryptone. It should be notedthat it is the first record showing that casamino acid in-creases the maximum production of amylase in similarspecies. However, bacto liver and ammonium sulphateinhibited the α-amylase production in Anoxybacillus sp.KP1. Saxena et al. (2007) also reported that ammonium

Fig. 5. Effect of carbon sources on the α-amylase production.

424 F. Matpan Bekler & K. Guven

Fig. 6. Effect of nitrogen sources on the α-amylase production.

Fig. 7. Effect of various starches and raw starches on the α-amylase production. The abbreviations are as follows: ss: soluble starch,ps: potato starch, ws: wheat starch, rs: rice starch, wrs: wheat raw starch, crs: corn raw starch, rrs: rice raw starch.

salts had an inhibitory effect on the amylase produc-tion.

Effect of various starches and raw starches on the α-amylase productionThe results in Figure 7 display the reducing sugar pro-duction from the hydrolysis of the various starches andraw starches at 50◦C by the enzyme obtained fromAnoxybacillus sp. KP1. The highest amount of reducingsugars was liberated from 2% soluble starch (10837.7

U/mL) followed by 1% potato starch (9782.3 U/mL),while the production of reducing sugars from the wheatstarch, rice starch, corn raw starch, rice raw starch,and wheat raw starch were low in comparison with con-trol. Malhotra et al. (2000), Ul-Haq et al. (2003) andParakash et al. (2009) similarly found that the produc-tion of α-amylase was higher in the presence of solu-ble starch, and it was greatly inhibited in the presenceof wheat starch. Konsula & Liakopoulou-Kyriakides(2007) found that the extracellular α-amylase produc-

Thermostable α-amylase from Anoxybacillus sp. KP1 425

Fig. 8. Effect of pH on activity and stability of the Anoxybacillussp. KP1 α-amylase.

tion decreased in the presence of wheat flour.

Characterisation of the partially purified amylolytic en-zymeThe amylolytic enzyme was partially purified by 80 %ammonium sulphate saturation. Enzyme activity stain-ing (zymogram) performed using the native PAGE(Yang et al. 2004) revealed one major stained band ap-pearing after staining. It was found that mainly solublestarch is hydrolysed, but not pullulan as the substrate.

Effect of pH on the activity and stability of the α-amylaseThe effect of pH on the α-amylase activity was stud-ied using soluble starch as a substrate at various pHvalues at 55◦C. The pH activity profile of this enzymeis shown in Figure 8. The optimum pH was found tobe 8.0. Previous studies also reported similar optimalpH value of 8.0 for Anoxybacillus pushinoensis (Farah-mand et al. 2009) and both Anoxybacillus sp. DT3-1and Anoxybacillus sp. SK3-4 (Chai et al. 2012).The Anoxybacillus sp. KP1 α-amylase was very

stable in a pH range of 6.0–10.0 and retained around70% activity after 1 h of incubation at 50◦C. Being8.0 as the optimum pH, the activity decreased abovepH 10.0 (Fig. 8). Anoxybacillus beppuensis TSSC-1 α-amylase was stable at pH 7.0 (Kikani & Singh 2012). Itwas determined that the α-amylases from Anoxybacil-lus sp. DT3-1 and Anoxybacillus sp. SK3-4 were stablein the pH range of 6.0–9.0 (Chai et al. 2012).

Effect of temperature on the activity and stability of α-amylaseThe supernatant amylolytic activities were assayed atdifferent temperatures. The Anoxybacillus sp. KP1 α-amylase activity increased with temperature upto 60◦C.A reduction in the enzyme activity was observed attemperatures above 70◦C (Fig. 9). The optimum tem-perature for the Anoxybacillus sp. KP1 α-amylase wasfound to be 60◦C, which is comparable to those de-

Fig. 9. Effect of temperature on activity of the Anoxybacillussp.KP1 α-amylase.

Fig. 10. Effect of temperature on stability of the Anoxybacillussp.KP1 α-amylase.

scribed for other Anoxybacillus α-amylases (Poli et al.2006; Farahmand et al. 2009; Chai et al. 2012; Kikani &Singh 2012). These are the properties considered to bevery important for industrial starch liquefaction. Fromthese results, the enzyme seemed to have considerablethermostability, which can be favourable in industrialoperations for traditional brewing and food process-ing, where the temperatures for pasteurization couldbe used to inactivate the enzymes after fermentation(Stamford et al. 2001; Lui & Xu 2008).For the thermal stability estimations, after the pre-

incubation of the partially purified enzyme for 30 minat temperatures of 60, 65, 70, 75, and 80◦C, it was foundthat the Anoxybacillus sp. KP1 α-amylase could be ac-tive up to 70◦C (Fig. 10). Kikani & Singh (2012) havereported that the Anoxybacillus beppuensis TSSC-1 pu-rified α-amylase was highly stable for 24 h at 50◦C, 15 hat 70◦C and 6 h at 90◦C. Chai et al. (2012) determinedthat the α-amylase was relatively stable at 60–65◦Cfrom both Anoxybacillus sp. DT3-1 and Anoxybacillussp. SK3-4.

426 F. Matpan Bekler & K. Guven

Table 2. Effects of metal ions and enzyme inhibitors.

Effect Relative activity (%)

Control 100Ca2+ 68Mn2+ 123Cu2+ 104Hg2+ 0SDS 11

Effects of metal ions and enzyme inhibitorsThe effect of different metal ions on the activity of theα-amylase is shown in Table 2. The Anoxybacillus sp.KP1 α-amylase was activated by Cu2+ and Mn2+ (rel-ative activity, 104% and 123%, respectively), whereasCa2+ and SDS decreased the activity by 22% and 89%,respectively. The unenhanced activity in the presence ofCa2+ was also confirmed for calcium-independent amy-lases reported previously by Kikani & Singh (2012) forAnoxybacillus beppuensis TSSC-1. In our study, the α-amylase activity was strongly inhibited in the presenceof Hg2+. Konsula & Liakopoulou-Kyriakides (2004),Najafi et al. (2005), Behal et al. (2006), Asgher et al.(2007) and Hmidet et al. (2008) stated that a strongerinhibitory effect was observed in the presence of Hg2+.The inhibition by Hg2+ may indicate the importanceof indole amino acid residues in enzyme function, ashas been demonstrated for other microbial α-amylases(Gupta et al. 2003). Furthermore, Hg2+ is known to oxi-dize indole rings and to interact with the aromatic ringspresent in tryptophan residues (Lui et al. 2010; Sharma& Satyanarayana 2012). It was also found that 1% SDSinhibited the amylase activity by 89%. Aygan et al.(2008) reported that SDS was the most effective effec-tor that shows the proportion of the hydrophobic aminoacid composition of the enzyme. Chai et al. (2012) de-termined that the α-amylase activity was strongly in-hibited in the presence of 1% SDS.

ConclusionIn this study, an isolate from Kopru, a hot-spring reser-voir in Diyadin in Turkey, was characterized as a mem-ber of Anoxybacillus genus using 16S rRNA sequencesas well as biochemical and physiological characteris-tics. This moderately thermophilic and alkaliphilic bac-terium named Anoxybacillus sp. KP1 (GenBank Ac-cession No.: KC525949) produces extracellular ther-mostable α-amylase. There are several reports on α-amylase from Anoxybacillus species. The studied en-zyme was found to be economical and stable at highertemperature which has the potential for being used inmany commercial applications, such as starch, food, de-tergent and pharmaceutical industries.

References

Apar K.D. & Ozbek B. 2004. α-Amylase inactivation by temper-ature during starch hydrolysis. Process Biochem. 39: 1137–1144.

Asgher A.M., Asad J.A.M., Rahman B.S.U. & Legge R.L. 2007.A thermostable α-amylase from a moderately thermophilicBacillus subtilis strain for starch processing. J. Food. Eng.79: 950–955.

Atanassova M., Derekova A., Mandeva R., Sjøholm C. & Kam-bourova M. 2008. Anoxybacillus bogrovensis sp. nov., a novelthermophilic bacterium isolated from a hot spring in DolniBogrov, Bulgaria. Int. J. Syst. Evol. Microbiol. 58: 2359–2362.

Aygan A., Arikan B., Korkmaz H., Dincer S.& Colak O.2008. Highly thermostable and alkaline α-amylase from ahalotolerant-alkaliphilic Bacillus sp. AB68. Braz. J. Micro-biol. 39: 547–553.

Behal A., Singh J., Sharma M.K., Puri P. & Batra N. 2006. Char-acterization of alkaline α-amylase from Bacillus sp. AB 04.Int. J. Agric. Biol. 8: 80–83.

Belduz A.O., Dulger S. & Demirbag Z. 2003. Anoxybacillus go-nensis sp. nov., a moderately thermophilic, xylose-utilizing,endospore forming bacterium. Int. J. Syst. Evol. Microbiol.53: 1315–1320.

Benson D.A., Cavanaugh M., Clark K., Karsch-Mizrachi I., Lip-man D.J., Ostell J. & Sayers E.W. 2013. GenBank. NucleicAcids Res. 41: D36–D42.

Bernfeld P. 1955. Amylases, α and β. Methods Enzymol. 1: 149–158.

Chai Y.Y., Abd Rahman R.N.Z.R., Illias R.M. & Goh K.M. 2012.Cloning and characterization of two new thermostable andalkalitolerant α-amylases from the Anoxybacillus species thatproduce high levels of maltose. J. Ind. Microbiol. Biotechnol.39: 731–741.

De Clerck E., Rodriguez-Diaz M., Vanhoutte T., Heyrman J.,Logan N.A. & De Vos P. 2004. Anoxybacillus contaminanssp. nov. and Bacillus gelatini sp. nov., isolated from con-taminated gelatin batches. Int. J. Syst. Evol. Microbiol. 54:941–946.

Derekova A., Sjøholm C., Mandeva R. & Kambourova M. 2007.Anoxybacillus rupiensis sp. nov., a novel thermophilic bac-terium isolated from Rupi Basin (Bulgaria). Extremophiles11: 577–583.

Dulger S., Demirbag Z. & Belduz A.O. 2004. Anoxybacillus ay-derensis sp. nov. and Anoxybacillus kestanbolensis sp. nov.Int. J. Syst. Evol. Microbiol. 54: 1499–1503.

Farahmand M., Mozafari N.A., Mehrabian S. & KhavarinejadR.A. 2009. Characterization of α-amylase produced by ther-mophilic bacteria isolated from Iranian hot spring waters. Pa-jouhesh Va Sazandegi 21: 161–167.

Gul-Guven R., Guven K., Poli A. & Nicolaus B. 2008. Anoxy-bacillus kamchatkensis subsp. asaccharedens subsp. nov., athermophilic bacterium isolated from a hot spring in Bat-man. J. Gen. Appl. Microbiol. 54: 327–334.

Gupta R., Gigras P., Mohapatra H., Goswami V.K. & ChauhanB. 2003. Microbial α-amylase: a biotechnological perspective.Process Biochem. 38: 1599–1616.

Goh K.M., Kahar U.M., Chai Y.Y., Chong C.S., Chai K.P., Ran-jani V., Illias R.M. & Chan K.G. 2013. Recent discoveriesand applications ofAnoxybacillus. Appl. Microbiol. Biotech-nol. 97: 1475–1488.

Haki G.D. & Rakshit S.K. 2003. Developments in industrially im-portant thermostable enzymes a review. Bioresource Technol.89: 17–34.

Hmidet N., Bayoudh A., Berrin J.G., Kanoun S., Juge N. &Nasri M. 2008. Purification and biochemical characteriza-tion of a novel α-amylase from Bacillus licheniformis NH1.Cloning, nucleotide sequence and expression of amyN gene inEscherichia coli. Process Biochem. 43: 499–510.

Kikani B.A. & Singh S.P. 2012. The stability and thermodynamicparameters of a very thermostable and calcium-independentα-amylase from a newly isolated bacterium, Anoxybacillusbeppuensis TSSC-1. Process Biochem. 47: 1791–1798.

Konsoula Z. & Liakopoulou-Kyriakides M. 2004. Hydrolysis ofstarches by the action of α-amylase from Bacillus subtilis.Process Biochem. 39: 1745–1749.

Konsula Z. & Liakopoulou-Kyriakides M. 2007. Co-production ofα-amylase and β-galactosidase by Bacillus subtilis in complexorganic substrates. Bioresource Technol. 98: 150–157.

Thermostable α-amylase from Anoxybacillus sp. KP1 427

Kumagai Y., Satoh T., Inoue A. & Ojima T. 2013. Enzy-matic properties and primary structures of two α-amylaseisozymes from the Pacific abalone Haliotis discus hannai.Comp. Biochem. Physiol. B Biochem. Mol. Biol. 164: 80–88.

Liu W., Shi P., Chen Q., Yang P., Wang G., Wang Y., Luo H.& Yao B. 2010. Gene cloning, over-expression, and charac-terization of a xylanase from Penicillium sp. CGMCC 1699.Appl. Biochem. Biotechnol. 162: 1–12.

Liu X.D. & Xu Y. 2008. A novel raw starch digesting α-amylasefrom a newly isolated Bacillus sp. YX-1: purification andcharacterization. Bioresource Technol. 99: 4315–4320.

Lowry O.H., Rosebrough N.J., Farr A.L. & Randall R.J. 1951.Protein measurement with the folin-phenol reagent. J. Biol.Chem. 193: 265–275.

Malhotra R., Noorwes S.M. & Satyanarayana T. 2000. Productionand partial characterization of thermostable and calcium-independent α-amylase of an extreme thermophilic Bacillusthermooleovorans NP54. Lett. Appl. Microbiol. 31: 378–384.

Najafi M.F., Deobagkar D. & Deobagkar D. 2005. Purifcation andcharacterization of an extracellular α-amylase from Bacillussubtilis AX20. Protein Expr. Purif. 41: 349–354.

Nascimento W.C. & Martins M.L.L. 2003. Production and prop-erties of an extracellular protease from thermophilic Bacillussp. Braz. J. Microbiol. 35: 91–96.

Prakash B., Vidyasagar M., Madhukumar S., Muralikrishna G.& Sreeramulu K. 2009. Production, purification, characteriza-tion of two extremely halotolerant, thermostable, and alkali-stable α-amylases from Chromohalobacter sp. TVSP 101.Process Biochem. 44: 210–215.

Pikuta E., Lysenko A., Chuvilskaya N., Mendrock U., HippeH., Suzina N., Nikitin D., Osipov G. & Laurinavichius K.2000. Anoxybacillus pushchinensis gen. nov., sp. nov., a novelanaerobic, alkaliphilic, moderately thermophilic bacteriumfrom manure, and description of Anoxybacillus flavithermscomb. nov. Int. J. Syst. Evol. Microbiol. 50: 2109–2117.

Poli A., Esposito E., Lama L., Orlando P., Nicolaus G., Appolo-nia F.D., Gambacorta A. & Nicolausa B. 2006. Anoxybacil-lus amylolyticus sp. nov., a thermophilic amylase producingbacterium isolated from Mount Rittmann (Antarctica). Syst.Appl. Microbiol. 29: 300–307.

Qader S.A., Bano S., Aman A., Syed N. & Azhar A. 2006. En-hanced production and extracellular activity of commerciallyimportant amylolytic enzyme by a newly isolated strain ofBacillus sp. AS-1. Turk. J. Biochem. 31: 135–140.

Saxena R.K., Dutt K., Agarwal L. & Nayyar P. 2007. A highlythermostable and alkaline amylase from a Bacillus sp. PN5.Bioresource Technol. 98: 260–265.

Stamford T.L.M., Stamford N.P., Coelho L.C.B.B. & Araujo J.M.2001. Production and characterization of a thermostable α-amylase from Nocardiopsis sp. endophyte of yam bean. Biore-source Technol. 76: 137–141.

Topal S., Pembeci C., Borcakli M., Batum M. & Celtik O. 2000.Investigation of selected industrially important enzymatic ac-tivities of Turkish agricultural mycoflora – I: amylase, lipase,protease. Turk. J. Biol. 24: 79–93.

Ul-Haq I., Ashraf H., Iqbal J. & Qadeer M.A. 2003. Productionof α-amylase by Bacillus licheniformis using an economicalmedium. Bioresource Technol. 87: 57–61.

Wind R.D., Buitelaar R.M., Eggink G., Huizing H.J. & Di-jkhuizen L. 1994. Characterization of a new Bacillus stearo-thermophilus isolate: a highly thermostable amylase-produ-cing strain. Appl. Microbiol. Biotechnol. 41: 155–162.

Yang S.J., Lee H.S., Park C.S., Kim Y.R., Moon T.W. & ParkK.H. 2004. Enzymatic analysis of an amylolytic enzyme fromthe hyper-thermophilic archaeon Pyrococcus furiosus revealsits novel catalytic properties as both an α-amylase and acyclodextrin-hydrolyzing enzyme. Appl. Environ. Microbiol.70: 5988–5995.

Yang X., Zhang F., Ren W., Nie G., Li S., Ming H., Tang S.,Zhang L. & Li W. 2012. A study on the best conditionfor thermostable α-amylase fermented by response surfacemethodology by strain Anoxybacillus sp.YIM 342. J. YunnanUniversity 4: 490–496.

Yumoto I., Hirota K., Kawahara T., Nodasaka Y., OkuyamaH., Matsuyama H., Yokota Y., Nakajima K. & Hoshino T.2004. Anoxybacillus voinovskiensis sp. nov., a moderatelythermophilic bacterium from a hot spring in Kamchatka. Int.J. Syst. Evol. Microbiol. 54: 1239–1242.

Received April 23, 2013Accepted January 6, 2014