Biologia 70/7: 853—862, 2015 Section Cellular and Molecular Biology DOI: 10.1515/biolog-2015-0111 Anoxybacillus sp. AH1, an α-amylase-producing thermophilic bacterium isolated from Darge¸cit hot spring ¨ Omer Acer 1 *, Hem¸ se Pırınc ¸c ¸ıo˘ glu 1 , Fatma Matpan Bekler 1 , Reyhan G¨ ul-G¨ uven 2 & Kemal G¨ uven 1 1 Dicle University, Science Faculty, Department of Biology, Molecular Biology Division, 21280 Diyarbakır, Turkey; e-mail: oacer21@gmail.com 2 Dicle University, Ziya G¨okalp Education Faculty, Science Teaching Division, 21280 Diyarbakır, Turkey Abstract: The present study was conducted to isolate α-amylase-producing thermophilic bacteria from Darge¸ cit hot springs in Turkey. The morphological, biochemical and physiological characterisation, as well as genetic analysis by 16S rRNA sequences indicated that the isolated strain AH1 was a member of Anoxybacillus genus. The strain was aerobe, Gram-positive and spore-forming rod, exhibiting optimum growth temperature and pH of 60 ◦ C and 7.0–7.5, respectively. Optimization of growth medium and enzyme assay conditions for extracellular α-amylase production by the novel thermophilic Anoxybacillus sp. AH1 were carried out in many different media containing a variety of carbon and nitrogen sources. Among various carbon and nitrogen sources, peptone (2054.1 U/mL) at 1% and maltose (1862.9 U/mL) at 0.5% increased α-amylase activity, compared to controls. Moreover, a high enzyme production was observed with potato starch at 0.5% and 1% (2668.4 U/mL and 3627 U/mL, respectively), as well as with 1% soluble starch (2051.9 U/mL). The enzyme activity was found to be rather high in the presence of CaCl2 up to 100 mM. Key words: Anoxybacillus; isolation and characterisation; α-amylase; thermophilic bacteria. Abbreviations: BM, basal medium; LB, Luria broth; NB, nutrient broth; TLC, thin-layer chromatography. Introduction The Bacillaceae are a family of Gram-positive rod- or coccus-shaped bacteria most of which produce endospores. Anoxybacillus flavithermus was formerly known as Bacillus flavothermus. The strain was firstly discovered in a New Zealand hot spring and was char- acterized as a yellow-pigmented facultative anaerobe, which grew in a temperature range of 30–70 ◦ C (Heinen 1982). The name Anoxybacillus was proposed based on its anaerobic property, living in anoxic condition where the dissolved oxygen is depleted. Pikuta et al. (2000) firstly described the genus Anoxybacillus. Currently, a number of species belongs to this genus (over twenty species, two subspecies), in- cluding Anoxybacillus flavithermus (Pikuta et al. 2000), Anoxybacillus gonensis (Belduz et al. 2003), Anoxy- bacillus pushchinoensis (Pikuta et al. 2003), Anoxy- bacillus ayderensis and Anoxybacillus kestanbolensis (Dulger et al. 2004), Anoxybacillus voinovskiensis (Yu- moto et al. 2004), Anoxybacillus contaminans (De Clerck et al. 2004), Anoxybacillus kamchatkensis (Kev- brin et al. 2005), Anoxybacillus amylolyticus (Poli et al. 2006), Anoxybacillus rupiensis (Derekova et al. 2007), Anoxybacillus thermarum (Poli et al. 2009), Anoxy- bacillus eryuanensis and Anoxybacillus tengchongensis (Zhang et al. 2010), Anoxybacillus salavatliensis (Ci- han et al. 2011), Anoxybacillus suryakundensis (Deep et al. 2013), Anoxybacillus kaynarcensis (Inan et al. 2013) and some subspecies, e.g. Anoxybacillus kamchatken- sis subsp. asacchardens subsp. nov. (Gul-G¨ uven et al. 2008). The Anoxybacillus species clustered together (92-99% 16S rRNA sequence similarity) and their near- est neighbours are found to be Geobacillus, Saccharo- coccus, and Aeribacillus in the range of 90–95% sim- ilarity (Deep et al. 2013; Goh et al. 2013). The com- plete genome sequence of the thermophilic A. flavither- mus strain WK1 (GenBank accession No.: CP000922) has been determined and comparative genome analy- sis indicates an extensive gene loss in the Geobacil- lus/Anoxybacillus branch following their divergence from other bacilli (Saw et al. 2008). The amylases can be obtained from a variety of or- ganisms, including plants, animals and microbes (Khan & Priya 2011). Among bacteria, Bacillus sp. is widely used in industrial applications for its thermostable α- amylase (Devi et al. 2012). However, there are only few studies on α-amylase-producing thermophilic Anoxy- bacillus species (Chai et al. 2012b; Goh et al. 2013). The major advantages of using microorganisms for * Corresponding author c 2015 Institute of Molecular Biology, Slovak Academy of Sciences