J. Microbiol. Biotechnol. (2013), 23(4), 489–498 http://dx.doi.org/10.4014/jmb.1211.11027 First published online January 25, 2013 pISSN 1017-7825 eISSN 1738-8872 A New Raw-Starch-Digesting α-Amylase: Production Under Solid-State Fermentation on Crude Millet and Biochemical Characterization Maktouf, Sameh 1,2,3,4 , Amel Kamoun 5 , Claire Moulis 2,3,4 , Magali Remaud-Simeon 2,3,4 , Dhouha Ghribi 1 , and Semia Ellouz Châabouni 1 * 1 Université de Sfax, ENIS, Unité Enzymes et Bioconversion, Route de Soukra km 4, 3038 Sfax, Tunisia 2 Université de Toulouse; INSA, UPS, INP; LISBP, 135 Avenue de Rangueil, F-31077 Toulouse, France 3 INRA, UMR792, Ingénierie des Systèmes Biologiques et des Procédés, F-31400 Toulouse, France 4 CNRS, UMR5504, F-31400 Toulouse, France 5 Université de Sfax, ENIS, Laboratoire de Chimie Industrielle II, Route de Soukra km 4, 3038 Sfax, Tunisia Received: November 13, 2012 / Revised: December 13, 2012 / Accepted: December 14, 2012 A new Bacillus strain degrading starch, named Bacillus sp. UEB-S, was isolated from a southern Tunisian area. Amylase production using solid-state fermentation on millet, an inexpensive and available agro-resource, was investigated. Response surface methodology was applied to establish the relationship between enzyme production and four variables: inoculum size, moisture-to-millet ratio, temperature, and fermentation duration. The maximum enzyme activity recovered was 680 U/g of dry substrate when using 1.38 × 10 9 CFU/g as inoculation level, 5.6:1 (ml/g) as moisture ratio (86%), for 4 days of cultivation at 37 o C, which was in perfect agreement with the predicted model value. Amylase was purified by Q-Sepharose anion-exchange and Sephacryl S-200 gel filtration chromatography with a 14-fold increase in specific activity. Its molecular mass was estimated at 130 kDa. The enzyme showed maximal activity at pH 5 and 70 o C, and efficiently hydrolyzed starch to yield glucose and maltose as end products. The enzyme proved its efficiency for digesting raw cereal below gelatinization temperature and, hence, its potentiality to be used in industrial processes. Key words: Bacillus sp., amylase, solid-state fermentation, millet, characterization α-Amylases (E.C. 3.2.1.1) hydrolyze α-1-4-glycosidic bonds in starch, glycogen, and other related carbohydrates starch to yield diverse products including dextrins and progressively smaller polymers of glucosyl units. Being one of the major industrial enzymes with approximately 25% of the enzyme market, amylases have found numerous applications in the food, chemical, textile, laundry, baking, and energy sectors [21, 32]. Amylases are commonly produced under submerged conditions, but solid-state fermentation (SSF) is often employed to produce larger amounts [37]. Many agro- industrial residues such as wheat bran, coconut oil cake, rice, sweet potato, or sweet sorghum have been used as supporting insoluble materials for bacterial or fungal SSF [28, 38, 43]. For optimal usage, these matrixes should provide all the needs for growth without supply of additional nutrients [7, 37]. Among agricultural crops, pearl millet is an attractive matrix for SSF. This insoluble starchy substrate, usually used in animal feed, has recently been shown to efficiently promote growth of several types of organisms in semi-SSF systems, in which additional nutrient sources were supplied for growth. For instance, Haq et al. [23] reported that addition of pearl millet to a rich medium containing other starch resources at 1% (w/v) allowed doubling of the amylase production level by Bacillus licheniformis. However, raw pearl millet has never been tested as a unique source of nutrients for the production of starch-degrading enzymes from bacterial or fungal origin. Owing to the expanding development of the starch industry, there is still a demand for enzymes produced at a low cost with improved properties such as high efficiency, Ca 2+ independency, stability, and raw starch digestibility [25]. In recent years, worldwide interest has been focused on the raw-starch-digesting amylases, which would be of value to simplify the starch process conversion, and to reduce the resulting production cost [20]. It was reported that fungi such as Aspergillus sp. [37] and Bacillus sp. were good producers of raw-starch- digesting amylases [36, 40]. *Corresponding author Phone: +216 74675331; Fax: +216 74275595; E-mail: semia.chaabouni@enis.rnu.tn