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

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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 2.0 M 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 4.19 Mb 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.

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Acknowledgements

We thank Maria Dolores Sotelo for her efforts to clone the α-amylase gene from G. dipsosauri and Drs. Pamela Marshall and Jeffrey Newman for facilitating the sequencing of the G. dipsosauri genome at Indiana University.

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Authors and Affiliations

  1. Microbion Research, 8931 W. Deanna Dr., Peoria, AZ, 85382, USA

    Charles E. Deutch

  2. School of Mathematical and Natural Sciences, Arizona State University, Glendale, AZ, 85306, USA

    Charles E. Deutch

  3. Bioinformatics Core Facility, Knowledge Enterprise, Arizona State University, Tempe, AZ, 85281, USA

    Shanshan Yang

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  1. Charles E. Deutch
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Charles E. Deutch did all of the microbiological and biochemical experiments and wrote the manuscript. Shanshan Yang did the assembly and analysis of the Gracilibacillus dipsosauri genome.

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Correspondence to Charles E. Deutch.

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Fig. S1

Multiple sequence alignment of the α-amylases from G. dipsosauri (PWU69658.1), G. halophilus (N4WMC6), G. orientalis (A0A1H9MMU5), G. kekensis (A0A1M7K410), and G. ureilyticus (A0A1I4H7R9). Fully conserved residues are indicated by (*); similar residues are indicated by (:) or (.) (PNG 146 kb)

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Deutch, C.E., Yang, S. Genomic sequencing of Gracilibacillus dipsosauri reveals key properties of a salt-tolerant α-amylase. Antonie van Leeuwenhoek 113, 1049–1059 (2020). https://doi.org/10.1007/s10482-020-01417-2

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