Identification of an Amylomaltase from the Halophilic Archaeon Haloquadratum walsbyi by Functional Metagenomics: Structural and Functional Insights

Abstract

Amylomaltases are prokaryotic 4-α-glucanotransferases of the GH77 family. Thanks to the ability to modify starch, they constitute a group of enzymes of great interest for biotechnological applications. In this work we report the identification, by means of a functional metagenomics screening of the crystallization waters of the saltern of Margherita di Savoia (Italy), of an amylomaltase gene from the halophilic archaeon Haloquadratum walsbyi, and its expression in Escherichia coli cells. Sequence analysis indicated that the gene has specific insertions yet unknown in homologous genes in prokaryotes, and present only in amylomaltase genes identified in the genomes of other H. walsbyi strains. The gene is not part of any operon involved in the metabolism of maltooligosaccharides or glycogen, as it has been found in bacteria, making it impossible currently to assign a precise role to the encoded enzyme. Sequence analysis of the H. walsbyi amylomaltase and 3D modelling showed a common structure with homologous enzymes characterized in mesophilic and thermophilic bacteria. The recombinant H. walsbyi enzyme showed starch transglycosylation activity over a wide range of NaCl concentrations, with maltotriose as the best acceptor substrate compared to other maltooligosaccharides. This is the first study of an amylomaltase from a halophilic microorganism.

Production of Large-Ring Cyclodextrins by Amylomaltases

“…This AM could efficiently exhibit starch transglycosylation activity in broad range of NaCl concentrations. This discovery might be promising for modifying starch in high ionic strength reaction and a direction for improving ionic strength tolerance of other AMs [18].…”

“…The optimum temperatures of characterized AMs are commonly around 60-70 • C for thermostable AMs and 30-45 • C for AMs from the mesophiles and plants. Halophilic AM was reported to exhibit optimal temperature at 40 • C [18]. However, some AMs from extreme thermophilic bacteria and archaea demonstrated higher optimum temperature such as the archaea Pyrobaculum aerophilum and Pyrobaculum calidifontis, with high disproportionation activity at 95 • C and 80 • C, respectively [11,23,24].…”