Halophiles and their enzymes: negativity put to good use
Introduction
Halophiles thrive from sea salinity (∼0.6 M) up to saturation salinity (>5 M NaCl), and include Archaea, Bacteria, and Eukarya [1]. Many halophilic microorganisms have been isolated from diverse environments, ranging from artificial solar salterns, to natural brines in coastal and submarine pools, and deep salt mines. Some of the most commonly observed halophiles are those flourishing in salterns used for salt production, e.g. Halobacterium spp. (a misnomer, being members of the domain Archaea), Salinibacter ruber (a member of the Bacteroidetes phylum), and Dunaliella salina (green alga of the Chlorophyceae class) (Table 1). Halophilic microorganisms also have long been recognized as agents of spoilage of fish and meat preserved with solar salt and some varieties have been used for fermentation of protein-rich foods.
Over the past few decades, adaptation of halophilic microorganisms to their environment has been the subject of increasing interest, with methodology for culturing, manipulation, and genetic engineering steadily advancing. Our understanding of the adaptation of halophiles to high salinity includes several different mechanisms for balancing the osmotic stress of the external medium. Halophilic Archaea (Haloarchaea) primarily use a ‘salt-in’ strategy, accumulating concentrations of KCl equal to NaCl in their environment, and where examined, their enzymes tolerate or require 4–5 M salt [2]. In contrast, most halophilic Bacteria and Eukarya, largely use a ‘salt-out’ strategy, excluding salts and accumulating or synthesizing de novo compatible solutes (e.g. glycine betaine and other zwitterionic compounds for Bacteria, and glycerol and other polyols for Eukarya) [3]. Among some halophiles, a combination of adaptive mechanisms may operate.
Early microbiologists addressing the adaptation of halophilic enzymes to high salinity discovered two primary features: a substantial number of protein charges and increased hydrophobicity [4]. Dissolved ions shielded electrostatic repulsions at low (<1 M) concentrations of salts and increased hydrophobic effects occurred at higher concentrations, from 4 M to saturating conditions. Roles for specific ion pairs were also sometimes suggested, e.g. in stabilizing active site regions or promoting subunit interactions. The combined effects of these forces were hypothesized to result in improved function in hypersaline conditions, where most non-halophilic proteins are inactivated by low water activity and limiting solvation, resulting in their denaturation, aggregation, and precipitation.
In the 1990s, the availability of the first solved structure of a halophilic enzyme and a halophile genome sequence provided a much more detailed molecular perspective on halophilic adaptations than previously available [5, 6, 7]. Subsequently, over the next two decades, there has been a veritable explosion in studies of halophiles and their enzymes [8]. In this article, we review the key features of halophilic proteins and enzymes revealed from bioinformatic, structural, genetic, and biochemical studies over the past few years and address some potential applications to biotechnology.
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Insights from bioinformatic analysis
The striking negativity of the halophilic proteome was first revealed by genome sequencing of Halobacterium sp. NRC-1 (Table 1) [6, 7, 8, 9]. A unimodal distribution of protein isoelectric points (pI) with a mean of 5.0 and mode of 4.2 was observed, in stark contrast to all non-halophilic proteomes which possess bimodal distribution with acidic and basic proteins and an average pI very close to neutrality (Figure 1). Halobacterium exhibited an excess of acidic (glutamic and aspartic acid) and a
Structural and biochemical characteristics
Structural and biochemical characterization of several halophilic enzymes has shown that enhancing solvation is the key requirement essential for maintaining solubility and activity of halophilic enzymes in low water activity, which can approach values as low as 0.75 in a saturated NaCl solution (Table 2) [8]. Under these extremely water-limited conditions, hydrogen bonds between negatively charged side chains and water molecules become critical to maintaining a stable hydration shell [5, 23].
Halophilic transformations for biotechnology
Properties of halophilic microorganisms and their negatively charged enzymes make them potentially very useful for biotechnology. Hypersaline brines in which halophiles flourish provide ideal conditions for carrying out many biotechnological transformations, due to their great abundance and exclusion of non-halophilic contaminants. Halophiles may serve as a source of many unique biomolecules, such as stable enzymes, biopolymers, and compatible solutes, and they may also be valuable for
Conclusion
It is anticipated that halophiles and their negatively charged enzymes will be put to good use and be of increasing value in future. Halophiles produce extraordinarily stable enzymes which function under conditions where conventional enzymes cease to function, denature, and precipitate. A host of recent studies have illuminated how halophilic enzymes manage to bind water tightly and maintain solvation and solubility in extremely high salinity and low water activity conditions. While future
References and recommended reading
• of special interest
•• of outstanding interest
Acknowledgements
Work in the authors’ laboratory is supported by National Institutes of Health grant AI107634 and National Aeronautical and Space Administration grant NNX10AP47G and by gifts to the Haloarchaeal Education & Research Development (HERD) fund administered by the University of Maryland Baltimore Foundation.
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