Genomic sequencing of Gracilibacillus dipsosauri reveals key properties of a salt-tolerant α-amylase.

Abstract

Gracilibacillus dipsosauri is a moderately-halophilic Gram-positive bacterium which forms an extracellular α-amylase that is induced by starch, repressed by D-glucose, and active in 2.0M KCl. Previous studies showed that while enzyme activity could be measured with the synthetic substrate 2-chloro-4-nitrophenyl-α-D-maltotrioside (CNPG3), other assays were inconsistent and the protein showed aberrant mobility during nondenaturing gel electrophoresis. To clarify the properties of this enzyme, the genome of G. dipsosauri was sequenced and was found to be 4.19Mb in size with an overall G+C content of 36.9%. A gene encoding an α-amylase composed of 691 amino acids was identified. The protein was a member of the glycosyl hydrolase 13 family, which had a molecular mass of 77,396 daltons and a pI of 4.39 due to an unusually large number of aspartate and glutamate residues (95/691 or 13.7%). BLAST analysis of the amino acid sequence revealed significant matches to other proteins with cyclodextrin glycosyltransferase activity. Partial purification of the protein from G. dipsosauri showed that fractions catalyzing the hydrolysis of CNPG3 and p-nitrophenyl-D-maltoheptoside also catalyzed the formation of β-cyclodextrin but not α-cyclodextrin or γ-cyclodextrin. Formation of β-cyclodextrin was not stimulated by high salt concentrations but did occur with rice, potato, wheat, and corn starches and amylopectin. These studies explain the unusual features of the α-amylase from G. dipsosauri and indicate it should be classified as EC 2.4.1.19. The availability of the complete genomic sequence of G. dipsosauri will provide the basis for studies on other enzymes from this halophile which may be useful for biotechnology.