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IN ARTICLE PRESS Bioresource Technology xxx (2007) xxxxxx Highly thermostable, thermophilic, alkaline, SDS and chelator resistant amylase from a thermophilic Bacillus sp. isolate A3-15 Burhan Arikan * Art and Science Faculty, Department of Biology Molecular and Microbiology Laboratory, University of Cukurova, 01330 Adana, Turkey Received 29 March 2006; received in revised form 1 June 2007; accepted 1 June 2007 Abstract A thermostable alkaline a-amylase producing Bacillus sp. A3-15 was isolated from compost samples. There was a slight variation in amylase synthesis within the pH range 6.0 and 12.0 with an optimum pH of 8.5 (8 mm zone diameter in agar medium) on starch agar medium. Analyses of the enzyme for molecular mass and amylolytic activity were carried out by starch SDSPAGE electrophoresis, which revealed two independent bands (86,000 and 60,500 Da). Enzyme synthesis occurred at temperatures between 25 and 65 C with an optimum of 60 C on petri dishes. The partial purication enzyme showed optimum activity at pH 11.0 and 70 C. The enzyme was highly active (95%) in alkaline range of pH (10.011.5), and it was almost completely active up to 100 C with 96% of the original activity remaining after heat treatment at 100 C for 30 min. Enzyme activity was enhanced in the presence of 5 mM CaCl2 (130%) and inhibition with 5 mM by ZnCl2, NaCl, Nasulphide, EDTA, PMSF (3 mM), Urea (8 M) and SDS (1%) was obtained 18%, 20%, 36%, 5%, 10%, 80% and 18%, respectively. The enzyme was stable approximately 70% at pH 10.011.0 and 60 C for 24 h. So our result showed that the enzyme was both, highly thermostable-alkaline, thermophile and chelator resistant. The A3-15 amylase enzyme may be suitable in liquefaction of starch in high temperature, in detergent and textile industries and in other industrial applications. 2007 Elsevier Ltd. All rights reserved. Keywords: Thermophile Bacillus sp.; a-Amylase; Highly thermostable; Alkaliphilic; Chelator resistant 1. Introduction Thermophilic microorganisms are adapted to thrive at temperatures above 60 C. They are a source of interesting enzymes that are both thermoactive and thermostable (Niehaus et al., 1999). The enzymes that have been isolated recently from these eotic microorganisms show unique features, are extremely thermostable and usually resistant against chemical denaturants such as detergents, chaotrophic agents, organic solvents and extremes of pH (Jorgensen et al., 1977). Amylases are among the most important enzymes and are of great signicance in present-day biotechnology. * Tel.: +90 322 3386084x2571; fax: +90 322 3386070. E-mail addresses: firstname.lastname@example.org, email@example.com Although they can be derived from several sources, such as plants, animals and microorganisms; enzymes from microbial sources generally meet industrial demands. The spectrum of amylase application has widened in many other elds, such as clinical, medical and analytical chemistries, as well as their widespread application in starch saccharication and in the textile, food, brewing and distilling industries (Pandey et al., 2000). Because of the industrial importance of amylases, there is ongoing interest in the isolation of new bacterial strains producing amylases suitable to new industrial applications, such as alkaline amylase for the detergent industry (McTigue et al., 1995). Amylases constitute a class of industrial enzymes having approximately 25% of the enzyme market (Rao et al., 1998). It is desirable that a-amylases should be active at the high temperatures of gelatinization (100110 C) and 0960-8524/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2007.06.019 Please cite this article in press as: Arikan, B., Highly thermostable, thermophilic, alkaline, SDS and chelator ..., Bioresour. Technol. (2007), doi:10.1016/j.biortech.2007.06.019 ARTICLE IN PRESS 2 B. Arikan / Bioresource Technology xxx (2007) xxxxxx liquefaction (8090 C) to economize processes; therefore, there has been a need and continual search for more thermophile and thermostable a-amylases (Sidhu et al., 1997). Several Bacillus sp. and thermostable Actinomycetes including Thermomonospora and Thermoactinomyces are versatile producers of the enzymes (Ben et al., 1999). The genus Bacillus produces a large variety of extracellular enzymes of which amylases and proteases are of signicant industrial importance. An highly thermostable and alkaline a-amylases is available from the mesophile Bacillus sp. PN5 (Saxena et al., 2007). The thermophilic bacterium B. stearothermophylus oers an alternative for commercial production of thermostable a-amylases. The advantages of using thermostable amylases in industrial processes include the decreased risk of contamination and cost of external cooling, a better solubility of substrates, a lower viscosity allowing accelerated mixing and pumping (Lin et al., 1998). The development of saccharifying amylolytic enzymes that are active at high temperatures (90 C) would directly benet the starch-processing industries. However, running a-amylase production processes at higher temperatures will require new process design and improved knowledge of thermophilic bacteria (Leveque et al., 2000). Processes using thermophiles still lack the maturity of classical processes with mesophilic bacteria and yeasts (Coolbear et al., 1992). Alkaliphilic Bacillus strains often produce enzymes active at alkaline pH, including alkaline a-amylase, protease and carboxymethylcellulase (Horikoshi, 1996). This article deals with the partial purication and some properties of the amylase produced by thermophile and alkaliphilic Bacillus sp. isolate A3-15 and their applications such as, new detergent formulation, textile, starch liquefaction and other industrial area. 2. Methods 2.1. Microorganism and culture conditions Bacillus sp. A3-15 was isolated from poultry manure compost samples collected in Adana, Turkey. To select of the gram-positive spore-forming bacteria Bacillus sp., compost was pasteurised at 80 C for 10 min (Hamilton et al., 1999). A3-15 was maintained and grown as described by McTigue (McTigue et al., 1995). The identication of isolated bacteria was various morphological and biochemical tests (Sodhi et al., 2005). This organism was found to produce an amylase on agar plates, a media composed with Na2HPO4 6 g, KH2PO4 3 g, NaCl 0.5 g, MgSO4 0.24 g, CaCl2 0.01 g, peptone 3 g, 1% (wt/vol) soluble starch (Merck), and Agar 15 g (Burhan et al., 2003). The initial pH was adjusted with NaOH to pH 9.0 after autoclaving (Milner et al., 1997). The organism was propagated at different temperatures (2065 C) and pHs (6.012.0). Amylase production was detected after ooding the plates with iodine solution (Burhan et al., 2003; Saxena et al., 2007). 2.2. Enzyme production The organisms were propagated at 60 C for 2 days in 100 ml of an M9 minimal medium, containing 1% soluble starch (Merck), placed in 1000 ml asks, with shaking on a shaker (250 rpm/min). The initial pH of the medium was about 9.0. After removal of cells by centrifugation (10,000 rpm, 20 min) at 4 C, the supernatant was used for partial purication (McTigue et al., 1995). 2.3. Partial purication of amylase Bacillus sp. A3-15 strain was grown 2 days. Cells were removed by centrifugation at 8000 rpm for 20 min at 4 C. The clear supernatant was concentrated (250 ml), with ethanol previously chilled to 20 C was added drop wise at 4 C with continuous stirring of 75%, and solution was left at 20 C for 24 h. The precipitate was recovered by centrifugation at 13,000 g for 20 min at 4 C. It was then resuspended in a minimum of phosphate (100 mM) at pH 7.0 (McTigue et al., 1995; Burhan et al., 2003). 2.4. Enzyme assay The relative amylase activity was assayed by adding 0.5 ml of enzyme to 0.5 ml soluble starch (1% v/v) in 100 mM BoraxNaOH buer pH 11.0, and incubating at 60 C for 60 min. The reaction was stopped by the addition of 2 ml of 3,5-dinitrosalicylic acid reagents and A550nm was measured in a Cecil 5500 spectrophotometer. One unit of amylase activity was dened as the amount of enzyme that released one micromole of reducing sugar equivalent to glucose per minute under the assay condition (Bernfeld, 1955). 2.5. Eects of pH and temperature on activity and stability The optimal temperature for activity was determined by assaying activity between 60 and 100 C for 60 min (Egas et al., 1998; Lo et al., 2001). Enzyme of pH optima, pH and heat stability were assayed under standard conditions at optimum enzyme temperature. The eect of pH on amylase activity was performed at 70 C in 100 mM Naphosphate buer (pH 6.58.0), GlycineNaOH buer (pH 8.5 10.5) and BoraxNaOH buer (pH 11.013.0) for 60 min, respectively (McTigue et al., 1995). For the measurement of pH stability, the enzyme was pre-incubated at 60 C for 24 h at pH 9.0, 10.0 and 11.0 in buer solutions. The residual activity was determined under the standard condition at optimum temperature for 60 min. Thermostability of the amylase was performed by enzyme samples at optimum pH for 30 min pre-incubating at temperatures between 60 and 100 C. The remaining activity was determined by incubating enzymes at optimum temperature for 60 min (Egas et al., 1998). Please cite this article in press as: Arikan, B., Highly thermostable, thermophilic, alkaline, SDS and chelator ..., Bioresour. Technol. (2007), doi:10.1016/j.biortech.2007.06.019 ARTICLE IN PRESS B. Arikan / Bioresource Technology xxx (2007) xxxxxx 3 2.6. SDSPAGE zymogram For determination of molecular weight, enzyme preparations and known molecular weight markers were subjected to electrophoresis with the use of homogenized 10% acrylamide gel. 0.2% soluble starch was incorporated into the separating gel prior to the addition of ammonium persulphate and polymerisation. After the electrophoresis, the gel was stained for 1 h with Comassie Blue R 250 dye in methanolacetic acidwater solution (4:1:5, by volume) and destained in the same solution without dye (Maniatis et al., 1982; Bollag et al., 1996). For zymogram of amylase activity, SDS was removed by washing the gel at room temperature in solutions containing 50 mM Na2HP04, 50 mM NaH2P04 (pH 7.2), isopropanol 40% for 1 h and 50 mM Na2HP04, 50 mM Na2HP04 (pH 7.2) for 1 h, respectively. Renaturation of enzyme proteins was carried out by keeping the gel overnight in a solution containing 50 mM Na2HP04, 50 mM Na2HP04 (pH 7.2), 5 mM b-mercaptoethanol and 1 mM EDTA at 4 C. Gel was stained in a solution of iodine (Iodine 5 g/l, KI 50 g/l), for 30 min, clear bands indicate the presence of amylase activity (Lee et al., 1994; Lin et al., 1998). The molecular mass of the enzyme was nally estimated from the position of standard proteins (Sigma M3788, 36,000, 45,000, 55,000, 66,000, 84,000, 97,000, 116,000 and 205,000 Da). 2.7. Eect of metal ions and chelating agent The eect of metal ions on amylolytic activity was determined by adding of dierent concentrations of each ion to the standard assay. All metals were used in the chloride such as EDTA (5 mM), CaCl (5 mM), NaCl (5 mM), Nasulphite (5 mM), ZnCl2 (5 mM), PMSF (3 mM), Urea (8 M) and SDS (1%). The activity of the enzyme alone in BoraxNaOH buer (pH 11.0) was taken to be 100%. The eect of metal ions, surfactants and chelating agent on amylolytic activity was determined by pre-incubating the enzyme in the presence of inhibitor for 30 min at 60 C, and then performing the assay in the presence of the same inhibitor concentration at optimum temperature for 60 min (Egas et al., 1998; Lo et al., 2001). 3. Results The isolated Bacillus sp. A3-15 from poultry manure was gram positive, rod shaped, aerobic, catalase positive and spore forming. According to the basis of various morphological and biochemical characteristic, it was identied as Bacillus sp. Enzyme synthesis of Bacillus sp. A3-15 occurred at temperatures between 25 and 65 C with an optimum of 60 C, while A3-15 Bacillus sp. grew well at between 20 and 65 C on starch agar medium. There was a slight variation in amylase synthesis within the pH range 6.0 and 11.0 with an optimum pH 8.5 on starch agar medium in petri dishes. The optimum temperatures for amylase production and growth of bacteria were obtained at 60 C and 2065 C, respectively. According to the paper chromatography results, the enzyme was determined as aamylase. 3.1. Molecular weight Molecular weight was determined by SDSPAGE electrophoresis as described in Section 2. Analyses of the enzyme by SDSPAGE revealed two bands that show amylolytic activity in starch gel. The molecular weight of these bands was estimated as 86 and 60.5 kDa (Fig. 1). 3.2. Properties of the partially puried a-amylase For estimation of the optimum temperature of the enzyme, the activity was determined at dierent temperatures (60100 C). The enzyme has a broad temperature range between 60 and 100 C and the optimum temperature was observed to be around 70 C. The activity of enzyme at the temperatures 60 C, 70 C, 80 C, 90 C and 100 C was as 99%, 100%, 91%, 87.5% and 60%, respectively (Fig. 2). The enzyme was highly active over 94% between the temperatures 6090 C. Experiments were repeated three times and mean values used. The pH optima were determined in three buer systems. The enzyme showed good activity at alkali pH. The optimum activity pH of the enzyme was 11.0. The optimal activity was around 89% between 9.5 and 12.5 pH ranges and especially pH 10.012.0 with an average 94% activity (Fig. 3). The pH stability of enzyme was determined by pre-incubating at 60 C for 24 h at three dierent pHs (pH 9.0, 10.0 and 11.0) and the relative activity was measured by the standard assay method. The enzyme showed about 75% (at pH 9.0), 67% (at pH 10.0) and 65% (at Fig. 1. Analysis of A3-15 a-amylase by 10% homogenized SDSPAGE. The samples were subjected to SDSPAGE and the gel was stained with iodine. Lanes (1) 15 ll, (2) 20 ll, (3) 25 ll, (4) 25 ll of A3-15 a-amylase and (5) Marker: (Sigma 3788, 36,000, 45,000, 55,000, 66,000, 84,000, 97,000, 116,000 and 205,000 Da). Please cite this article in press as: Arikan, B., Highly thermostable, thermophilic, alkaline, SDS and chelator ..., Bioresour. Technol. (2007), doi:10.1016/j.biortech.2007.06.019 ARTICLE IN PRESS 4 105 100 Relative enzyme activity (%) 95 90 85 80 75 70 65 60 55 50 60 70 80 T e mperature C 90 100 B. Arikan / Bioresource Technology xxx (2007) xxxxxx 101 Retain relative enzyme activity (%) 100 99 98 97 96 95 94 Cont 60 70 80 90 Pre-incubated temperatures C 100 Fig. 4. Eect of temperature on thermal stability of Bacillus sp. A3-15 aamylase. Fig. 2. Eect of temperature on the activity and stability of Bacillus sp. A3-15 a-amylase. The enzyme displayed maximal activity at 70 C. Table 1 Eect of dierent chemical sources with dierent concentrations on the activity of a-amylase from Bacillus sp. A3-15 Eectors Control EDTA CaCl2 ZnCl2 NaCl Nasulphite SDS Urea PMSF Concentration None 5 mM 5 mM 5 mM 5 mM 5 mM 1% 8M 3 mM Remaining enzyme activity (%) 100 95.0 130 82.0 80.0 64.0 82.0 20.0 90.0 110 100 Relative enzyme activity (%) 90 80 70 60 50 40 30 6.5 7 7.5 8 8.5 9 9.5 10 10.5 11 11.5 12 12.5 pH Fig. 3. Eect of pH on the activity of Bacillus sp. A3-15 a-amylase. The maximal enzyme activity was obtained at pH 11.0. pH 11.0) remaining activity after treatment under standard enzyme assay condition. For thermal stability estimations, after pre-incubation of the enzyme for 30 min at temperatures, of 60 C, 70 C, 80 C, 90 C and 100 C, the original activity retained was 100%, 99%, 98%, 98%, and 96%, respectively. The enzyme was completely active up to 100 C, with 96% of the original remaining activity after heat treatment at 100 C for 30 min (Fig. 4). 3.3. Eect of dierent metal ions, chelator, surfactants and inhibitors The eect of dierent metal ions and inhibitors were investigated on partially puried enzyme pre-incubated at 60 C for 30 min. The residual activity was measured at optimum pH and temperature. The activity of the enzyme alone in BoraxNaOH (pH 11.0) buer was taken to be 100%. Among the tested eectors, urea (8 M) inhibited activity up to 80%. The denaturation of the original enzyme activity with urea (80%) supports to that the enzyme consists of hydrophobic amino acid composition. When the A3-15 amylase enzyme was incubated with EDTA (5 mM), NaCl (5 mM), Nasulphite (5 mM), ZnCl2 (5 mM), PMSF (3 mM) and SDS (1%), the enzyme activity was retained at 95%, 80%, 64%, 82%, 90% and 82% of the original activity. EDTA generally shows non-competitive inhibition of amylase activity and a slight inhibition showed us it is a metallo enzyme. On the other hand, a stimulated activity was observed in the presence of Ca2+ (5 mM) around 130% from the original activity (Table 1). The highly increase activity in the presence of Ca2+ also showed that the enzyme has a rigid structure. 4. Discussion In industry, bacterial a-amylases are produced mainly from cultures of Bacillus subtilis var. amyloliquefaciens. (Uhlig, 1998; Goyal et al., 2005). Bacillus stearotermophilus and Bacillus licheniformis a-amylases are well characterized and are heavily used in the starch-processing industry. Since, thermostability is an important feature for use of amylolytic enzymes in starch-processing, amylases from thermophilic and hyperthermophilic bacteria are of special interest as a source of novel thermostable enzymes (Leveque et al., 2000; Saxena et al., 2007). Please cite this article in press as: Arikan, B., Highly thermostable, thermophilic, alkaline, SDS and chelator ..., Bioresour. Technol. (2007), doi:10.1016/j.biortech.2007.06.019 ARTICLE IN PRESS B. Arikan / Bioresource Technology xxx (2007) xxxxxx 5 The optimal temperature for amylase production and growth of the Bacillus sp. A3-15 strain were found to be dierent as the organism has growth and production optima at 60 C on petri dishes. Similar observations were also reported for Bacillus sp. PN5 (Saxena et al., 2007). According to the zymogram analyses of A3-15 a-amylase enzyme was determined that consisting of two subunits as 86 kDa and 60.5 kDa. While bigger subunit, 86 kDa, showed higher activity, small unit, 60.5 kDa, showed lesser activity (Fig. 1). On pH optimum analyses, A3-15 a-amylase produced two peaks at pH 8.0 and 11.0. However, the optimum enzyme activity was also detected at pH 11.0. These results were supported that, 86 kDa subunit was showed an optimum activity at pH 11.0 and the subunit 60.5 kDa was showed an optimum activity at pH 8.0. Multiple amylase production has also been reported from many Bacillus strains (1989; Hagihara et al., 2001). In literatures were reported that the two enzyme referring 76 and 53 kDa from thermophilic Bacillus, while two independent band with 150 and 42 kDa from thermophilic and alkaliphilic Bacillus sp. TS-23 (Lin et al., 1998; Mamo and Gessesse, 1999). It has been reported that the alkaline amylase of Bacillus sp. Ant-6 was alkaline, thermophile and thermostable (Burhan et al., 2003). Bacillus sp. A3-15 amylase enzyme results showed that the optimum temperature and pH were 70 C and 11.0, respectively (Figs. 2 and 3). The enzyme was highly active at pH range of 10.011.5 (95%). The enzyme was stable between pH ranges 10.011.0 for 24 h (70%). These ndings were highly similar with the literature results (Mamo and Gessesse, 1999; Horikoshi, 1999; Goyal et al., 2005). These results was showed that, A3-15 amylase enzyme highly alkaline, pH stable and thermophile. While the optimal temperature and pH for activity 70 C and 9.0, respectively from amylase of Bacillus sp. TS-23 (Lin et al., 1998), The temperature prole our enzyme showed that the enzyme had an optimum of 70 C with 60% activity retained 100 C at pH 11.0. Mamo and Gessesse (1999) also reported optimal temperature of 75 80 C for amylase from Bacillus sp. WN11. While Horikoshi (1999) observed temperature optimum for the activity of a-amylase was at 70 C. Our results showed that A3-15 a-amylase enzyme had nearly 100% activity temperature 60100 C for 30 min. It was also retaining 96% original activity at 100 C, pH 11.0. Similarly 100% activity at 90 C for 1 h for amylase from Bacillus sp. has been reported by Teodoro and Martins (2000). A novel strain of Bacillus stearothermophilus was isolated from the samples of a potato processing industry, which has a highly thermostable amylase. In fact, a-amylases from Bacillus genus is heat stable and may be used starch hydrolysis up to 90 C. Most other thermophile Bacillus amylases reported to so far, two amylases exhibited higher temperature optimum for activity and showed good thermal stability (Brawn and Kelly, 1993; Dong et al., 1997; Horikoshi, 1999). These are the properties considered to be very important for industrial starch liquefaction. Although most a-amylases are inhibited by metal ions, alkaline amylases vary in their response to the chelator, EDTA with some being unaected (McTigue et al., 1995; Jana et al., 1997). In the presence of 1 mM EDTA, Amy-K38 retained full activity ion in the presence of as high as 100 mM (Hagihara et al., 2001), while Egas et al. (1998) reported 88% activity with 10 mM EDTA. A3-15 a-amylase has also slight inhibition by 5% with 5 mM EDTA. A3-15 a-amylase enzyme was strongly inactivated by 8 M Urea (80%). It was reported that amylases from alkaliphilic Bacillus strains were not inhibited by 10 mM EDTA at 30 C but was completely inactivated by 8 M urea (Horikoshi, 1999). These results show that the A3-15 a-amylase is an alkaliphilic enzyme. The eect of Zn2+ also varied between amylases. For instance, it had a potent inhibitory eect on the amylases from Schwanniomyces alluvius and Bacillus cereus NY 14, whereas it could have no eect at all on the enzyme of Aspergillus kowachii (Lin et al., 1998). As the thermostable a-amylases from a thermophilic Bacillus 46% (Lin et al., 1998) and 13% inhibition were reported suggesting that the inhibition with Zn2+ determines the thermostability of enzyme (Mamo and Gessesse, 1999). The inhibition of A3-15 (by 18%) in the presence of Zn2+ ion indicates that is thermostable enzyme. Similar results were found thermostable a-amylase from a thermophilic Bacillus for AmyII inhibition of 13% with zinc (Mamo and Gessesse, 1999). The thermophilic and alkaliphilic Bacillus sp. TS-23 enzyme activity remained up to 115% (Lin et al., 1998), while thermostable amylase activity from Bacillus stearothermophilus was retained up to 122% with 1 mM CaCl2 (Srivastava, 1987). Similarly A3-15 a-amylase enzyme showed an enzymatic activity around 130% in the presence of 5 mM CaCl2. The positive eects of Ca2+ ion presence on the thermostability enzymes have been shown including the amylases from B. Licheniformis, Pyrococcus juriosus and Thermococcus litoralis (Brawn and Kelly, 1993; Dong et al., 1997). A3-15 a-amylase has also slight inhibition by 5% with 3 mM PMSF and Lin et.al were also reported similar result (97% with 10 mM) (Lin et al., 1998). In order to have applications in detergent industries, amylase must be stable to various detergent ingredients, such as surfactants and chelators. The amylase from Bacillus sp. A3-15 exhibited more than 82% activity when preincubated with 1% SDS at 60 C for 30 min. Saxena et al. (2007) reported that, a highly thermostable and alkaline amylase enzyme, there was 86.36% stability after 1 h incubation with SDS. Our results was indicated that, A3-15 amylase enzyme highly stable with 1% SDS (82%). It was stated that LAMY was resistant to incubation at 40 C for 1 h with 0.1% SDS (Igarashi et al., 1998), additionally Lo et al., reported that SDS had no marked eect on enzyme activity ion as concentration 8% (Lo et al., 2001). This resistance, which is essential requirements, suggest that the enzyme may be used as an eective additive in Please cite this article in press as: Arikan, B., Highly thermostable, thermophilic, alkaline, SDS and chelator ..., Bioresour. Technol. (2007), doi:10.1016/j.biortech.2007.06.019 ARTICLE IN PRESS 6 B. Arikan / Bioresource Technology xxx (2007) xxxxxx Enzymatic properties of a novel liquefying a-amylase from an alkaliphilic Bacillus isolate and entire nucleotide and amino acid sequences. Applied and Environmental Microbiology 64 (9), 3382 3389. Jana, M., Chattopadhyay, D.J., Pati, B.R., 1997. Thermostable, high salttolerant amylase from Bacillus megaterium VUMB-109. Acta Microbiologica Immunologica Hungarica 44 (3), 281289. Jorgensen, S., Vorgias, C.E., Antranikian, G., 1977. Cloning sequencing and expression of an extracellular a-amylase from the hypertermophilic archeon Pyrococccus furiosus in Escherichia coli and Bacillus subtilis. Journal of Biological Chemistry 272, 1633516342. Lee, S., Morikawa, M., Takagi, M., Imanakawa, T., 1994. Cloning aapT Gene and Characterization of its product, a-amylase, pullulanase (aapT), from Thermophilic and alkaliphilic Bacillus sp. Strain XAL601. Applied Environmental Microbiology 60, 37613773. Leveque, E., Janacek, S., Haye, B., Belarbi, A., 2000. Thermophilic archeal...