Halophiles and their enzymes: negativity put to good use

https://doi.org/10.1016/j.mib.2015.05.009Get rights and content

Highlights

  • Halophiles produce stable enzymes that are active under high salt conditions.

  • Halophilic enzymes maintain solvation and solubility in low water activity.

  • Halophiles are valuable for industrial processes in organic solvents and brine.

  • Halophilic processes may be scaled up in absence of strictly sterile conditions.

Halophilic microorganisms possess stable enzymes that function in very high salinity, an extreme condition that leads to denaturation, aggregation, and precipitation of most other proteins. Genomic and structural analyses have established that the enzymes of halophilic Archaea and many halophilic Bacteria are negatively charged due to an excess of acidic over basic residues, and altered hydrophobicity, which enhance solubility and promote function in low water activity conditions. Here, we provide an update on recent bioinformatic analysis of predicted halophilic proteomes as well as experimental molecular studies on individual halophilic enzymes. Recent efforts on discovery and utilization of halophiles and their enzymes for biotechnology, including biofuel applications are also considered.

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.

Section snippets

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.

References (49)

  • M.F. Roberts

    Organic compatible solutes of halotolerant and halophilic microorganisms

    Saline Systems

    (2005)
  • S.T. Bayley et al.

    Recent developments in the molecular biology of extremely halophilic bacteria

    Crit Rev Microbiol

    (1978)
  • A. Dym et al.

    Structural features that stabilize halophilic malate dehydrogenase from an archaebacterium

    Science

    (1995)
  • W.-L. Ng et al.

    Snapshot of a large dynamic replicon from a halophilic archaeon: megaplasmid or minichromosome?

    Genome Res

    (1998)
  • W.V. Ng et al.

    Genome sequence of Halobacterium species NRC-1

    Proc Natl Acad Sci U S A

    (2000)
  • R. Karan et al.

    Function and biotechnology of extremophilic enzymes in low water activity

    Aquat Biosyst

    (2012)
  • S.P. Kennedy et al.

    Understanding the adaptation of Halobacterium species NRC-1 to its extreme environment through computational analysis of its genome sequence

    Genome Res

    (2001)
  • S. DasSarma et al.

    Post-genomics of the model haloarchaeon Halobacterium sp. NRC-1

    Saline Systems

    (2006)
  • A. Bolhuis et al.

    Halophilic adaptations of proteins

  • M.D. Capes et al.

    The core and unique proteins of haloarchaea

    BMC Genomics

    (2012)
  • S.L. DasSarma et al.

    HaloWeb: the haloarchaeal genomes database

    Saline Systems

    (2010)
  • N. Sharma et al.

    The halophile protein database

    Database

    (2014)
  • E.A. Becker et al.

    Phylogenetically driven sequencing of extremely halophilic archaea reveals strategies for static and dynamic osmo-response

    PLoS Genet

    (2014)
  • P.L. Kastritis et al.

    Haloadaptation: insights from comparative modeling studies of halophilic archaeal DHFRs

    Int J Biol Macromol

    (2007)
  • Cited by (202)

    • Marine enzymes: Classification and application in various industries

      2023, International Journal of Biological Macromolecules
    View all citing articles on Scopus
    View full text