ORIGINAL ARTICLE

Isolation of a thermophilic Anoxybacillus flavithermus sp.nov. and production of thermostable α-amylaseunder solid-state fermentation (SSF)

Sadin Özdemir & Fatma Matpan & Veysi Okumus &

Abdurrahman Dündar & Mehmet Sefa Ulutas &

Mert Kumru

Received: 28 December 2010 /Accepted: 8 November 2011 /Published online: 2 December 2011# Springer-Verlag and the University of Milan 2011

Abstract A new bacteria was isolated from hot-springwater of Gazlıgöl, Afyonkarahisar in Turkey. Based onmorphological and biochemical tests and 16S rRNA genesequence analysis, the isolate belonged to the Anoxybacillusflavithermus species which has 99% similarity with thebacterium DNA. The production of α-amylase bythermophilic Anoxybacillus flavithermus was investigatedunder solid-state fermentation by using some agriculturalwaste as substrates. Solid substrates such as rice husk,banana husk, millet, water melon husk, lentil bran, wheatbran and maize oil cake were studied for enzymeproduction. Of these, rice husk was proved as the bestsubstrate for α-amylase production (1,271 U/mg). Themaximum α-amylase production was observed as 1,803U/mg at 72 h, 1,000 μm particle size, 70% initial moisturecontent (w/v), and 40% inoculum level (v/w). Among thevarious nitrogen sources tested, 1% peptone (3,170 U/mg)was found to be the best nitrogen source for α-amylaseproduction. As additional carbon sources, 1% starch(2,364 U/mg) enhanced α-amylase production. The

optimum temperature for the activity of α-amylase wasfound to be 70°C. The enzyme was optimally active at pH6.0 and stable in the pH range of 6.0–8.0.

Keywords Agro-industrial .α-Amylase . Solid-statefermentation (SSF) . Thermophilic bacteria

Introduction

α-Amylase (EC 3.2.1.1) is an endo-acting hydrolase thathydrolyzes the internal α-1,4 linkages in starch in a randomfashion leading to the formation of soluble maltodextrins,maltose, and glucose (Gomes et al. 2003; Gangadharan etal. 2006). These enzymes have great significance withextensive biotechnological applications such as bread,baking, food, textile, paper industries, starch processing,and pharmacy (Pandey et al. 2000). α-Amylases are a classof industrial enzymes having approximately 25% of theenzyme market (Arıkan et al. 2003; Asgher et al. 2007).

Thermophilic Bacillus species, with optimal growthtemperature between 45 and 70°C, have been isolated froma wide range of environments and are important contami-nants of heat-treated food products. Research interest inthese organisms has increased due to their biotechnologicalpotential, especially as sources of thermostable enzymes(Studholme et al. 1999). Bacteria belonging to the genusBacillus have been widely used for commercial productionof thermostable α-amylase (Malhotra et al. 2000).Thermostable α-amylases are commercially important invarious starch-processing industries (Crabb and Mitchinson1997). The most widely used thermostable α-amylases areproduced by mesophilic and thermophilic microorganismswhich are considered to have an enormous potential as

S. Özdemir (*) :V. Okumus :A. Dündar :M. S. UlutasDepartment of Biology, Faculty of Arts and Science,Siirt University,56100 Siirt, Turkeye-mail: sadin77@hotmail.com

F. MatpanDepartment of Biology, Faculty of Arts and Science,Dicle University,21280 Diyarbakır, Turkey

M. KumruDepartment of Molecular Biologyand Genetic and Biotechnology, Faculty of Science and Letters,Istanbul Technical University,34469 Maslak, Istanbul, Turkey

Ann Microbiol (2012) 62:1367–1375DOI 10.1007/s13213-011-0385-4

sources of thermostable α-amylases of industrial importance(Mamo and Gessesse 1999). Babu and Satyanarayana(1995), have reported the suitability of thermophilicBacillus coagulans for α-amylase production by solid-statefermentation in flasks, reactor and trays.

Solid-state fermentation (SSF) technology has beenknown for centuries; from approximatetly 2600 BC, itwas used by the Egyptians for making bread andinformation on the ‘koji process’ that dates back to1000 BC. The koji process is of great historicalimportance to modern SSF technology and can beconsidered as the prototype of SSF (Ooijkaas et al.2000). SSF process is defined as the fermentationinvolving solids in the absence of free-flowing water;however, substrates must possess enough moisture tosupport growth and metabolism of the microorganisms(Ooijkaas et al. 2000; Pandey 2003).

The SSF technique is generally confined to processesinvolving fungi (Ul-Haq et al. 2002; Baysal et al. 2003).However, successful bacterial growth in SSF is known innatural fermentation. Bacillus species are considered to bethe most important sources of α-amylase and have beenused for enzyme production using SSF (Pandey et al. 2000;Anto et al. 2006; Ozdemir et al. 2009). This may be due totheir ability to adhere to the substrate particles, to producefilamentous cells for penetration into substrate particles andtheir requirement for higher water activity (Babu andSatranarayana 1996).

Traditionally, α-amylase has been produced by sub-merged fermentation (SmF) and used in a one-way processin solution (Kunamneni et al. 2005). The cost of enzymeproduction in SmF is high, which necessitates reduction inproduction cost by alternative methods. The contents ofsynthetic media are very expensive and these contentsmight be replaced with more economically availableagricultural by-products for the reduction of cost of themedium (Anto et al. 2006). In recent years, however, theSSF processes utilised have been more and more for theproduction of this enzyme. SSF is preferred to SmF becauseof superior productivity, simple technique, low capitalinvestment, low energy requirement, less water output,better product recovery, and lack of foam build up, and ithas been reported to be the most appropriate process fordeveloping countries (Tanyildizi et al. 2007). The use ofagricultural polymeric substrates like wheat, rice, soy,cassava, etc. (Sivaramakrishnan et al. 2006) makes SSFan attractive alternative method (Anto et al. 2006).

Thermophilic fermentations are considered quite usefulfor technical and environmental purposes. Advantages are,for instance, a reduction in cooling costs, a better solubilityof substrates, a lower viscosity allowing accelerated mixingand pumping, and a reduced risk of microbial contamina-tion (Wind et al. 1994).

In this study, morphological and biochemical tests and16S rRNA gene sequence analysis were performed.Different parameters such as solid substrates, incubationtime, particle size, initial moisture content of the substrate,and inoculum size were optimized for maximal α-amylaseproduction under SSF using agricultural waste as substrateby thermophilic Anoxybacillus flavithermus.

Materials and methods

Morphological and biochemical tests and 16S rRNA gene124 sequence analysis

In this study, a bacterium, which was isolated by Dr. SadinOzdemir from hot-spring water of Gazlıgöl, Afyonkarahisarin Turkey, was used. Tests for the morphological andbiochemical identification of the obtained isolate wereconducted. To determine the characteristics of the bacteriumGram, spore-staining methods and motility tests were used.Some characteristics of the isolate, such as starch, gelatine andcaseine hydrolysis, catalase, and lipase activities, weredetermined.

The bacterium was incubated at 60°C in a rotary shakerat 120 rpm for 16 h. Then, DNA isolation was performedby FastDNA Spin kit (MP, Biomedical, USA) protocol.

DNA isolation Protocol

Firstly, a 2-mL liquid bacterium sample was taken to alysing tube and centrifuged at 14,000 g for 1 min. Then,978 μL sodium phosphate buffers and 122 μL MT bufferwas added to the lysing tube. After that, the tube washomogenized in the FastPrep instrument for 40 s at thespeed setting of 6.0. The sample was centrifuged at14,000 g for 5 min and the supernatant was transferred toa clean 2.0 mL microcentrifuge tube. Then, 250 μL PPS(protein precipitation solution) was added to the tube. Thetube was shaken by hand 10 times. The sample wascentrifuged at 14,000 g for 5 min and the supernatant wastransferred to a clean 15-mL tube. Then, 1.0 mL bindingmatrix suspension was added to the 15-mL tube. To allowbinding of the DNA, the tube was inverted by hand for2 min. After that, the tube was placed in a rack for 3 min.Then, 500 μL supernatant was carefully removed. Thesample was resuspended in the remaining amount ofsupernatant and 600 μL of the mixture was added to aSPIN Filter tube and centrifuged at 14,000 g for 1 min. Thecatch tube liquid was removed and the remaining mixturewas added to the SPIN Filter tube and centrifuged at14,000 g for 1 min. Lastly, the catch tube liquid wasremoved again and 500 μL prepared SEWS-M (salt ethanol

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wash) solution was added to the tube and gently resus-pended. Then, the tube was centrifuged at 14,000 g for1 min and the catch tube liqid was removed. The tube wascentrifuged a second time without any addition at 14,000 gfor 2 min. The catch tube was removed and replaced by anew catch tube. The SPIN Filter tube was held for 5 min atroom temorature. After that, the binding matrix wasresuspended using 100 μL DES [DNA elution solution(DNase/Pyrogen-free water)]. In the final step, the tube wascentrifuged at 14,000 g for 1 min to bring the eluted DNAinto the clean catch tube. After that, the DNA was presentin the catch tube.

Isolated DNA was run in 1% (0.5 g agar/50 mL 1×TAEbuffers) agarose gel at 110 V and visualized under the GelDoc (Biorad, USA) imaging system. The 16S rRNA generegion was amplified by using 63f/1,387r primers fromisolated DNA. Those primers are shown in Table 1. PCRreaction conditions are shown in Table 2. The PCR productwas purified by using the QIAquick PCR Purification Kitprotocol. After that, sequence PCR was performed by usingthe 63f primer. Then, the PCR product was run in 1%agarose gel and visualized under the Gel Doc (Biorad)imaging system. After the DNA amount was measured withthe Qubit Florometre (İnvitrogen, USA), sequence purifi-cation was performed. Then, the DNA sample wassequenced with an Abi 3130 DNA Sequence Analyzer(Abi 3130). After the sequence of the 16S rRNA gene, thesequence was compared within GenBank (Benson et al.1999) using BLAST search (Altschul et al. 1997). Mostclosely related 16S rRNA genes of species were determinedand phylogenetic trees were constructed with the neighbor-joining method using the Molecular Evolutionary GeneticsAnalysis (MEGA) (Tamura et al. 2007).

Preparation of inoculum

The bacterial culture was grown in 20 mL of NutrientBroth (with tap water) taken in a 100-mL Erlenmeyerflask and inoculated with a loopfull of cells from a 24-h-old slant and kept at 60°C in a rotary shaker(120 rpm). After 10 h of incubation, 1 mL of thisculture was used as inoculum. By serial dilution andplating, the number of viable colonies in the inoculumwas found to be 3×108 CFU/mL.

Solid-state fermentation (SSF)

Rice husk (RH), banana husk (BH), millet (M), watermelon husk (WMH), lentil bran (LB), wheat bran (WB),and maize oil cake (MOC) were obtained from a localmarket in Diyarbakır, Turkey. These agro-industrials wereused as solid substrates and their effects on theproduction of α-amylase were determined. The bestsolid substrate for α-amylase was selected and used insubsequent experiments.

Three grams of dry substrates which passed throughsieve of 1,500 μm were placed into 100-mL Erlenmeyerflasks. To adjust moisture levels (% by mass per volume),0.1 M Tris HCI (pH 7.0) was added. After autoclaving at121°C for 15 min, and cooling to room temperature, theflasks were inoculated with 2 mL spore suspension andincubated at 60°C at 120 rpm.

Optimization of process parameters

The effect of various physico-chemical parameters requiredfor the optimum production of α-amylase by Anoxybacillusflavithermus was studied in SSF. The strategy was tooptimize each parameter independently and we employedoptimal conditions subsequently in all experiments.Incubation time (24–144 h), particle size (500–2,500 μm),initial moisture content of the substrate (40–80% by massper volume), and inoculum size (10–70% by mass pervolume) were optimized. Studies were also performed toevaluate the influence of different carbon sources (starch,glucose, galactose, sucrose, maltose, and lactose at 1% w/v)and nitrogen sources (yeast extract, tryptone, peptone, beefextract, ammonium sulphate, ammonium nitrate, andammonium chloride at 1% w/v) by adding them to thefermentation medium.

Enzyme extraction

The fermented substrates were mixed thoroughly with tapwater. The contents were mixed by shaker at 120 rpm. Theslurry was squeezed through a muslin cloth. The extract

Table 1 63f/1,387r primers which were used for amplifying the 16SrRNA gene region

Primername

Primer sequence Reference

Eu1387r 5’ GGGCGGWGTGTACAAGGC 3’ Lane et al. 1991

Eub63f 5’ CAGGCCTAACACATGCAAGTC 3’ Lane et al. 1991

63f/1,387r

Temperature(°C)

Time Cycles

94 3 min 1

94 30 s 3549 30 s

72 45 s

72 10 min 1

4 – 1

Table 2 PCR conditions for63f/1,387r primers in the PCRreaction

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was centrifuged at 10,000 g for 10 min and then the clearsupernatant used as the crude enzyme.

Enzyme assay

α-Amylase activity was determined according to theBernfeld method (1955). Amounts of 0.1 mL enzymesolution and 0.2 mL 0.5% starch solution in 0.1 M Tris–HCl buffer (pH 6.0) were incubated for 30 min at 70°C. Atthe end of this duration, the reaction was stopped byaddition of 0.4 mL DNS (3,5 dinitro salicylic acid) and keptin boiling water for 5 min. Then, it was diluted by 10 mLdistilled water, and spectrophotometric measurements wereconducted at 489 nm. One unit of enzyme activity wasdefined as the amount of protein that produced 1 mg ofreducing sugar per mL of enzyme solution in 30 min.

All the experiments were performed in triplicate and thestandard error was expressed.

Effect of temperature on crude enzyme activity and stability

The optimum enzyme temperature was evaluated bymeasuring the α-amylase activity at different temperatures(30–90°C) in 0.1 M citrate buffer (pH 6.0). The effect oftemperature on α-amylase stability was determined bymeasuring the residual activity after 15–120 min attemperatures ranging from 60 to 80°C. The residualenzyme activity was determined as described earlier.

Effect of pH on crude enzyme activity and stability

The optimum enzyme pH was determined with various pHin the reaction mixture by using the following buffers;citrate (0.1 M pH 3.0–6.0), Tris-HCI (0.1 M pH 7.0–9.0),and carbonate/bicarbonate (0.1 M pH 10.0). To determinethe stability of α-amylase, the enzyme was pre-incubated indifferent buffers (pH 3.0–10.0) for 60 min. The residualenzyme activity was determined as described earlier.

Results and discussion

Morphological and biochemical tests and 16S rRNA genesequence analysis

The morphological and biochemical features of the isolateare shown in Table 3. The isolate was Gram-positive andhad rod-shaped cells, the ability to form spores, and wasmotile.

The DNA was extracted from bacteria. The amount ofDNAwas measured as 0.09 μg/mL and the extraction resultwas visualized by GelDoc™ (Biorad) (Fig. 1a). In addition,the result of the sequence PCR was visualized by agarose

gel (Fig. 1b). Both isolation and sequence PCR resultsshowed successfully agarose gel profiles. The DNASequence Analyzer (Abi 3130) performed sequences about850 nucleotides of 16S rRNA gene from the bacteriumDNA. The sequence result is shown below;

GTGAGTAACACGTGGGCAACCTGGGGGGAGACGGGGATAACACCGAGAAATCGGTGCTAATACCGGATAACACGAAATGTCGCATGACGTTTCGTTGAAAGACGGCGCAAGCTGTCGCTACAGGATGGGCCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGCGAAGAAGGCCTTCGGGTCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTAACGTAGTAACTGGCGTTACTGTGACGGTACCTAACGAGAAAGCCACGGCTA

Characteristics Anoxybacillusflavithermus

Aerobic growth +

Gram painting +

Spore forming +

Cell form Rod-shaped

Pigmentation Yellow

Hemolysis −Motility +

Hydrolysis of:

Starch ++

Caseine +

Gelatine −Activity of:

Lipase +

Catalase +

Table 3 Morphological andbiochemical tests

+ Positive result, − negativeresult

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Fig. 1 Effect of different substrates on α-amylase production byBacillus sp. under solid-state fermentation. Process conditions:incubation time 48 h, inoculum size 20% (% by volume per mass),initial moisture levels 60% (% by volume per mass), particle size1,500 μm, temperature 60°C

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ACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGTTCCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGGGACTTGAGTGCAGAAGAGGAGAGCGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGGCTCTCTGGTCTGTAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTATCCACCCTTTAGTGCTGTAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGCTCGCAAGAGTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTT…..

The sequence was compared to related bacteria sequen-ces. The BLAST results showed that the most relatedspecies to the 16S rRNA gene region sequence wasAnoxybacillus flavithermus species which has 99% similar-ity to the bacterium DNA. Also, the phylogenetic tree wasconstructed in MEGA (Molecular Evolutionary GeneticsAnalysis) using the ten most related bacteria (Fig. 2)

Effect of different substrates on α-amylase production

α-Amylase production was optimized by using thermophilicAnoxybacillus flavithermus under SSF with different solidsubstrates such as RH, BH, M, WMH, LB, WB, and MOC.The study on the potential of different solid substratesfor α-amylase production showed RH (1,271 mg/U) asthe most effective substrate (Fig. 3). The SSF process issignificantly influenced by the nature of the solid substrate(Pandey 2003; Tanyildizi et al. 2007). In the SSF, solidmaterial is water insoluble polymers of cellulosic or starchmaterial that acts both as physical support and a source ofnutrients (Babu and Satranarayana 1996). RH has been

widely reported to be the best substrate for α-amylaseproduction in SSF due to its rich content of, e.g., vitaminsand minerals (Kunamneni et al. 2005). In subsequentexperiments, therefore, RH was used as the substrate forthe production of α-amylase.

Effect of incubation time on α-amylase production

The production of bacterial α-amylase by using the SSFtechnique requires less fermentation time, 24–48 h(Sivaramakrishnan et al. 2006). During the time coursestudy, enzyme activity was detected in the culture superna-tant from 24 h to 144 h, and the maximum α-amylaseproduction was found in 72 h (1,426 U/mg) (Fig. 4).Incubation beyond 72 h was undesirable as this resulted indecreased enzyme yields. The reason for this might havebeen due to the depletion of the nutrients or denaturation ofthe enzyme caused by the interaction with other componentsin the medium or a change in the pH of the medium (Mahantaet al. 2008). Optimized incubation time for α-amylase

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Fig. 3 Effect of inoculum size (% by volume per mass) on α-amylaseby Bacillus sp. under solid-state fermentation using RH as substrate.Process conditions: incubation time 72 h, initial moisture levels 60%(% by volume per mass), particle size 1,500 μm, temperature 60°C

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Fig. 2 Effect of incubation time on α-amylase production by Bacillussp. under solid-state fermentation using RH as substrate. Processconditions: inoculum size 20% (% by volume per mass), initialmoisture content 60% (% by volume per mass), particle size1,500 μm, temperature 60°C

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Fig. 4 Effect of initial moisture content (% by volume per mass)on α-amylase production by Bacillus sp. under solid-state fermentationusing RH as substrate. Process conditions: incubation time 72 h,inoculum size 40% (% by volume per mass), particle size 1,500 μm,temperature 60°C

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production by Aspergillus oryzae and Bacillus amylolique-faciens in SSF also showed maximum activities after 72 h(Ramachandran et al. 2004; Gangadharan et al. 2006). Thisincubation time has been used in later studies because of thehighest activity seen at 72 h.

Effect of inoculum size on α-amylase production

The inoculum level is also an important factor forproduction of α-amylase (Kunamneni et al. 2005). Theresults presented in Fig. 5 show that there was a significantincrease in α-amylase production with an increase ininoculum size up to an optimum level after which enzymeyield decreased. Maximum enzyme production wasobtained of 1,594 U/mg with 40% of inoculum size. Theproduction of α-amylase was very low at 10% of inoculumsize. The decrease seen with larger inoculum sizes could bedue to the shortage of nutrients available for the largerbiomass and faster growth culture (Sandhya et al. 2005).Therefore, 40% of inoculum size was used for furtherstudies.

Effect of initial moisture content of substrate on α-amylaseproduction

Substrate moisture is a crucial factor in SSF and itsimportance for enzyme production has been well estab-lished (Ramachandran et al. 2004). The moisture content ofthe medium changes during fermentation as a result ofevaporation and metabolic activities, and the moisture levelof the substrate is therefore most important (Baysal et al.2003). The critical importance of moisture level in SSFmedia and its influence on the biosynthesis and secretion ofenzymes can be attributed to the interference of moisture inthe physical properties of the solid particles (Kunamneni etal. 2005). Low and high moisture levels of the substrate

affect the growth of the microorganism resulting in lowerenzyme production (Sivaramakrishnan et al. 2006).

Figure 6 shows the effect of moisture content on enzymeproduction. Optimal enzyme production was found to be1,691 U/mg at 70% moisture contents of substrate. Earlierresearchers reported that moisture levels in SSF processesvaried between 30 and 85% (Gangadharan et al. 2006), andRamesh and Lonsane (1990) have also reported thatmaximum α-amylase production by Bacillus licheniformiswas found to be at 60–75% of moisture content.

Effect of substrate particle size (μm) on α-amylaseproduction

In the SSF processes, particle size is the most critical formicrobial growth and activity (Pandey 1991). The maxi-mum enzyme production (1,803 U/mg) was found to be at

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Fig. 6 Effect of carbon source (1%) supplementation on α-amylaseproduction by Bacillus sp. under solid-state fermentation using RH assubstrate. Process conditions: incubation time 72 h, inoculum size40% (% by volume per mass), initial moisture content 70% (% byvolume per mass), particle size 1,000 μm, temperature 60°C

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Fig. 5 Effect of substrate particle size (μm) on α-amylase productionby Bacillus sp. under solid-state fermentation using RH as substrate.Process conditions: incubation time 72 h, inoculum size 40% (% byvolume per mass), initial moisture content 70% (% by volume permass), temperature 60°C

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Fig. 7 Effect of nitrogen source (1%) supplementation on α-amylaseproduction by Bacillus subtilis under solid-state fermentation usingRH as substrate. Process conditions: incubation time 72 h, inoculumsize 40% (% by volume per mass), initial moisture content 70% (% byvolume per mass), particle size 1,000 μm, temperature 60°C

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1,000 μm particle size (Fig. 7). With smaller particles, thesurface area for growth is greater, and thus it should beconsidered as a desirable factor. However, too smallparticles may result in substrate agglomeration in mostcases, which interfere with aeration and may thus result inpoor growth. At the same time, larger particles providebetter aeration efficiency, but provide limited surface formicrobial attack (Elibol and Moreira 2005). Hence, it isstated that determination of particle size corresponding tooptimum growth and enzyme production is necessary(Sivaramakrishnan et al. 2006). Thus, 1,000 μm particlesize of substrate was used for further studies.

Effect of supplementation of RH with carbon sourceson α-amylase production

Simple and complex carbon sources, viz. starch, glucose,galactose, sucrose, maltose, and lactose, were used ascarbon sources to investigate their effect on α-amylaseproduction (Fig. 8). Maximal enzyme production was foundwith starch (2,369 U/mg) followed by maltose and sucrose.An extreme thermophile Bacillus thermoleovorans has

preferred starch and maltose as favorable carbon sourcesfor α-amylase production (Narang and Satyanarayana2001). Starch is known to increase enzyme production inBacillus sp. PS-7 and Bacillus sp. I-3 (Tanyildizi et al.2005, Goyal et al. 2005). Galactose and lactose had noeffect on α-amylase production. As indicated in Fig. 8,glucose showed a negative effect on α-amylase production.It has been reported that the synthesis of carbohydrate-degrading enzymes in most species of the genus Bacillus issubject to catabolic repression by glucose (Rajagopalan andKrishnan 2008a).

Effect of nitrogen source on α-amylase production

The influence of supplementary nitrogen sources such asyeast extract, peptone, tryptone, beef extract, ammoniumsulphate, ammonium nitrate, and ammonium chloride wasstudied. Added nitrogen sources have been reported to havean inducing effect on the production of various enzymesincluding α-amylase in the SSF system (Gangadharan et al.2006). Of the nitrogen sources tested, peptone increased theα-amylase production (3,168 U/mg) followed by yeastextract (3,021 U/mg) and beef extract (2,252 U/mg)

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Fig. 8 Effect of temperature on the activity of amylase at pH 6.0

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(Fig. 9). Peptone has been reported as a good nitrogensource for α-amylase production in Bacillus sp. andBacillus thermooleovorans (Tanyildizi et al. 2005;Malhotra et al. 2000). Rajagopalan and Krishnan (2008b))reported that yeast extract was the best nitrogen source forα-amylase production by B. subtilis KCC103. Among thenitrogen sources, ammonium sulphate, ammonium nitrate,and ammonium chloride were found to have no influenceon the production of α-amylase.

Effect of temperature on crude enzyme activity and stability

Optimum temperature of α-amylase of Anoxybacillusflavithermus was found to be 70°C (Fig. 10), which iscomparable to that described for other Bacillus α-amylases(Arıkan et al. 2003; Asgher et al. 2007). The effect oftemperature on stability was determined by incubating theenzymes at 60, 70 and 80°C for 15–120 min. α-Amylaseshowed good stability at 60, 70 and 80°C, maintaining 85,70 and 35%, respectively, of the original activity after 2 hof incubation for these temperatures (Fig. 11).

Effect of pH on crude enzyme activity and stability

The pH profil of the α-amylase was determined by assayingthe activity using different buffers of various pH values. α-Amylase was optimally active at pH 6.0, and 90% of thepeak activity was displayed in the pH range of 5.0–8.0.Optimum α-amylase activity has been found to be at pH6.0 by Bacillus sp. IMD 435, Bacillus sp. SA1, and B.subtilis US116 (Hamilton et al. 1999; Soni et al. 2003;Messaoud et al. 2004). The effect of pH on stability wasdetermined by incubating α-amylase at 37°C for 1 h atdifferent pH values. α-Amylase was stable in the pH rangeof 6.0–7.0. The enzyme retained 64 and 61% of itsmaximum activity at pH 4.0 and 9.0, respectively. Theuses of α-amylases that operate at lower pH values reducethe formation of unwanted products, such as maltulose,which are usually produced at high operation pH (Mamoand Gessesse 1999) Therefore, α-amylase of Anoxybacillusflavithermus that are optimally active and stable at pH 6.0may have a good potential to be used simultaneously forimproved starch hydrolysis.

In this study, a novel bacteria was isolated from hot-spring water of Gazlıgöl, Afyonkarahisar in Turkey. Basedon 16S rRNA gene region sequence, the microorganismwas closely related to Anoxybacillus flavithermus with 99%similarity. These studies showed that RH could be a goodsubstrate for α-amylase production by thermophilicAnoxybacillus flavithermus. Evidently, RH provided thenecessary nutrients for the microorganism to grow andsynthesize the enzyme. Supplementation carbon sources of1% starch and nitrogen sources 1% peptone and yeast

extract to the substrate positively enhanced the enzymesynthesis. In conclusion, because RH is an agriculturalwaste, α-amylase production in SSF by using RH assubstrate is both economical and ecologically important.

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