Cloning and characterization of two new thermostable and alkalitolerant α-amylases from the Anoxybacillus species that produce high levels of maltose

Abstract

Two genes that encode α-amylases from two Anoxybacillus species were cloned and expressed in Escherichia coli. The genes are 1,518 bp long and encode 506 amino acids. Both sequences are 98% similar but are distinct from other well-known α-amylases. Both of the recombinant enzymes, ASKA and ADTA, were purified using an α-CD-Sepharose column. They exhibited an optimum activity at 60°C and pH 8. Both amylases were stable at pH 6-10. At 60°C in the absence of Ca²⁺, negligible reduction in activity for up to 48 h was observed. The activity half-life at 65°C was 48 and 3 h for ASKA and ADTA, respectively. In the presence of Ca²⁺ ions, both amylases were highly stable for at least 48 h and had less than a 10% decrease in activity at 70°C. Both enzymes exhibited similar end-product profiles, and the predominant yield was maltose (69%) from starch hydrolysis. To the best of our knowledge, most α-amylases that produce high levels of maltose are active at an acidic to neutral pH. This is the first report of two thermostable, alkalitolerant recombinant α-amylases from Anoxybacillus that produce high levels of maltose and have an atypical protein sequence compared with known α-amylases.

Isolation and production of thermostable α-amylase from thermophilic Anoxybacillus sp. KP1 from Diyadin hot spring in Ağri, Turkey

“…• C, which is comparable to those de- scribed for other Anoxybacillus α-amylases (Poli et al 2006;Farahmand et al 2009;Chai et al 2012;Kikani & Singh 2012). These are the properties considered to be very important for industrial starch liquefaction.…”

“…In later studies, Belduz et al (2003) and Dulger et al (2004) Chai et al (2012) also isolated thermophilic Anoxybacillus sp. DT3-1 and SK3-4 from two hot springs in Malaysia.…”

“…Aygan et al (2008) reported that SDS was the most effective effector that shows the proportion of the hydrophobic amino acid composition of the enzyme. Chai et al (2012) determined that the α-amylase activity was strongly inhibited in the presence of 1% SDS.…”

Purification and Characterization of a Liquefying α-Amylase from Alkalophilic ThermophilicBacillussp. AAH-31

“…The resulting mutant enzyme was stable at 70 C at pH 8.5 for 10 min in the presence of 0.1 mM CaCl 2 , but its thermostability decreased significantly in the absence of the calcium ion. 13,14) Recently, alkalitolerantamylases, which were highly stable at 60 C in the absence of the calcium ion, were found in Anoxybacillus species, 15) but the enzymes predominantly produced maltose from starch even at the initial stage of the reaction, indicating that they are saccharifying enzymes, not suitable for use in detergents. In this study, we screened a thermostable alkaline liquefying -amylase with high resistance to chelating regents from soil bacteria, and successfully obtained an enzyme with the required properties from Bacillus sp.…”

Purification and Characterization of Thermostable and Detergent-Stable Alpha-Amylase from Anoxybacillus sp. AH1

“…Alpha-amylases (α-1,4-glucan-4-glucanohydrolase; EC 3.2.1.1) are a family of endoamylases that randomly cleave α-1,4-glucosidic linkages in starch and related carbohydrates to produce oligosaccharides of diff erent lengths and glucose in the α-anomeric form (1)(2)(3). α-Amylases can be obtained from diff erent sources, including plants, animals and microorganisms (4,5).…”