Purification and characterization of maltooligosaccharide-forming a-amylase from moderately halophilic Marinobacter sp. EMB8 Sumit Kumar, S.K. Khare ⇑ Department of Chemistry, Indian Institute of Technology, Delhi, New Delhi, India article info Article history: Available online 7 December 2011 Keywords: Halophiles a-Amylase Maltooligosaccharides Marinobacter sp. Solvent stable abstract Maltooligosaccharides especially maltotriose and maltotetraose producing amylases are highly desirable for application in bread making and other food industries. A maltotriose and maltotetraose producing amylase from moderately halophilic Marinobacter sp. EMB8 is described. Under optimized culture condi- tions, 48.0 IU/mL amylase was obtained. The enzyme was purified to homogeneity by ultrafiltration, DEAE cellulose and Sephadex G-75 column chromatography with 52% yield and 76-fold purification. It was a monomeric protein of 72 kDa. The amylase had many novel features viz. stability up to 20% NaCl, 80 °C temperature, pH 6.0–11.0 and in wide range of organic solvents at high concentrations. The enzyme efficiently hydrolyzed starch into maltooligosaccharides rich in maltotriose and maltotetraose. These novel properties make the Marinobacter sp. amylase a potentially useful enzyme. Ó 2011 Elsevier Ltd. All rights reserved. 1. Introduction Maltooligosaccharides (MOS) have important industrial applica- tions due to their high viscosity, water holding capacity and crystal- lization inhibition properties (Palacios et al., 2004). MOS with small oligosaccharides like maltotriose to maltopentaose prevent migra- tion of moisture from starch granules, reduce retrogradation by inhibiting realigning of amylose and amylopectin chains and inter- fere with starch–gluten interaction (Min et al., 1998; Nagarajan et al., 2006). This makes them quite useful as antistaling agent in the bread industries. For these reasons, there has been a growing interest in amylases that can produce MOS rich in maltotriose and maltotetraose. Amylases with ability to synthesize MOS from starch have been reported from Bacillus stearothermophilus US100 (Ali et al., 2001), Bacillus subtilis (Messaoud et al., 2004), Brachybacterium sp. strain LB25 (Doukyu et al., 2007) and Bacillus acidicola (Sharma and Satyanarayana, 2010). Only few strains viz. B. subtilis (Takasaki, 1985) and Bacillus sp. GM8901 (Kim et al., 1995), produce maltotri- ose and maltotetraose. Hence, search continues for efficient malto- triose/maltotetraose producing amylases. Oligosaccharide synthesis is favored in presence of organic sol- vents and at high temperature (Riva and Roda, 2000). Doukyu et al. (2007) have exploited the solvent stability of Brachybacterium sp. strain LB25 amylase, in DMSO to improve product selectivity. The present study aims at screening microbial strains producing sol- vent and heat stable amylase with maltotriose and maltotetraose forming ability. Halophiles have been considered as potential source of enzymes, stable in salt and organic solvents (Karan and Khare, 2010; Margesin and Schinner, 2001; Ventosa et al., 1998). Since they inhabit in high salt surrounding which reduces water activity significantly, their enzymes are attuned to function in low water medium and exhibit stability towards organic solvents (Le Borgne et al., 2008). The solvent stable amylases have been reported from Nesterenkonia sp. strain F (Shafiei et al., 2011) and Haloarcula sp. (Fukushima et al., 2005). The MOS forming ability has not been investigated in any of these cases. The present study describes a novel halophilic Marinobacter sp. EMB8 strain isolated from Kozhikode, India. The strain secretes a solvent and heat stable amylase. The production, purification, char- acterization of the amylase and its application in MOS synthesis are encompassed. 2. Methods 2.1. Microorganism Marinobacter sp. EMB8 was isolated from the sea coast of Kozhikode (Kerala, 11°25 0 N 75°77 0 E) by salt enrichment. It was re- lated to Marinobacter sp. by 16S rDNA sequence analysis and sub- mitted in GenBank, NCBI, USA with accession number GU059908. 2.2. Amylase production Amylase production was carried out under optimized condi- tions, viz. medium containing (g/L): starch, 50; casein enzyme 0960-8524/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2011.11.109 ⇑ Corresponding author. Address: Enzyme and Microbial Biochemistry Labora- tory, Department of Chemistry, Indian Institute of Technology, Delhi Hauz Khas, New Delhi 110016, India. Tel.: +91 11 2659 6533; fax: +91 11 2658 1102. E-mail addresses: skhare@rocketmail.com, skkhare@chemistry.iitd.ac.in (S.K. Khare). Bioresource Technology 116 (2012) 247–251 Contents lists available at SciVerse ScienceDirect Bioresource Technology journal homepage: www.elsevier.com/locate/biortech