Purification and characterization of a thermostable phytate resistant α-amylase from Geobacillus sp. LH8

Abstract

A thermophilic and amylolytic bacterium (LH8) was isolated from the hot spring of Larijan in Iran at 65 °C. Identification of strain LH8 by 16S rDNA sequence analysis showed that LH8 strain belongs to the Geobacillus sp. with 99% sequence similarity with the 16S rDNA of Geobacillus thermodenitrificans. A new α-amylase (GA) was extracted from this strain and purified by ion-exchange chromatography. SDS-PAGE showed a single band with an apparent molecular mass of 52 kDa. The optimum temperature and pH were 80 °C and 5–7, respectively. In the presence of Mn 2+, Ca 2+, K +, Cr 3+ and Al 3+, the enzyme activity was stimulated while Mg 2+, Ba 2+, Ni 2+, Zn 2+, Fe 3+, Cu 2+ and EDTA reduced the activity. The K m and V max values for starch were 3 mg ml −1 and 6.5 μmol min −1, respectively. The gene encoding α-amylase was isolated and the amino acid sequence was deduced. Comparison of GA and other α-amylase amino acid sequences suggested that GA has conserved regions that were previously identified in α-amylase family but GA exhibited some substitutions in the sequence. Its phytate resistant is an important property of this enzyme. 5 and 10 mM phytic acid did not inhibit this enzyme. Therefore, features of phytate resistant α-amylase from Geobacillus sp. LH8 are discussed.