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

Claudia Leoni,Hai Tran,Luigi R Ceci,Caterina Manzari,Graziano Pesole,Peter N Golyshin,Mariateresa Volpicella

doi:10.3390/life12010085

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.

Highlights

Recombinant enzymes have been extensively studied for their use in biocatalysis processes in the food, pharmaceutical, chemical, biofuels, and textile industries
In this study we report the identification, by means of a functional metagenomics screening of the saltern of Margherita di Savoia (Italy), of the gene for an amylomaltase from the halophilic archaeon H. walsbyi and the expression of the enzyme in E. coli cells
Water samples were collected from the crystallization pond “Imperatrice” (36% of salinity, 30 ◦C, pH 7.20) of the Margherita di Savoia saltern, located on the south-eastern coast of Italy (Lat. 41◦23 10” N, Long. 16◦7 14” E) on June 2017

Summary

Introduction

Recombinant enzymes have been extensively studied for their use in biocatalysis processes in the food, pharmaceutical, chemical, biofuels, and textile industries. Biotechnological studies have focused on enzymes obtained from extremophilic microorganisms, called extremozymes, which have high stability in extreme conditions of temperature or pH, in the presence of organic solvents and at high ionic concentrations. Starch is synthesized in plants as a heterogeneous compound, mainly containing the two polymers amylose and amylopectin. Amylopectin consists of α-1,4-polyglucoside molecules of 10–60 glucose units, containing α-1,6-branches of the α-1,4-polyglucosides of 15–45 glucose units. The side chains are further branched, giving rise to amylopectin molecules containing on average two million glucose units. Extremozymes active at low temperatures or high salt concentrations are being studied for possible applications on starch [13,14]

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