We have established that the glycoside hydrolase GH-42 family bga gene in the cold-adapted Antarctic haloarchaeon H. lacusprofundi produces a β-galactosidase protein that is polyextremophilic. In order to characterize the salient properties of this novel enzyme, we developed a cold-inducible, cold shock protein cspD2 gene promoter-based expression plasmid in the genetic model system, Halobacterium sp. NRC-1, and overexpressed the H. lacusprofundi bga gene. A high level of active β-galactosidase protein was produced in Halobacterium sp. NRC-1 and purified by gel filtration and hydrophobic interaction chromatography, and its identity was established by LC-MS/MS, SDS-PAGE, and ONPG hydrolysis. We found that the β-galactosidase enzyme was overexpressed 20-fold, and displayed very similar properties, with optimal activity at nearly saturated concentration of salts, 4 M NaCl or KCl, and significant measurable activity at low and even subzero temperatures, as well as temperatures above 50°C. Interestingly, we also found that the enzyme was active in the presence of 10–20% organic solvents, including methanol, ethanol, n-butanol, and isoamyl alcohol. All together, these findings show a remarkable β-galactosidase displaying enzyme activity at multiple extreme conditions, with significant potential for biotechnological applications
[8, 42–45]. The enzyme also serves as an excellent model for potential enzymatic activity in extraterrestrial conditions, such as those found on Mars
The H. lacusprofundi β-galactosidase is one of few polyextremophilic enzymes to be purified and studied in detail
. In the past, a barrier to such studies has been the requirement of high salt concentrations to obtain enzyme activity during overexpression in a foreign host, since low ionic strength conditions generally lead to misfolding or inactivation
[8, 47]. To avoid problems overproducing active H. lacusprofundi β-galactosidase in common non-halophilic hosts such as E. coli, we chose the haloarchaeal host, Halobacterium sp. NRC-1, for overexpression. This was anticipated to be an optimal host due to its high internal salt concentration, viability at low temperatures, completely sequenced genome, lack of endogenous β-galactosidase, and many available microbiological and molecular genetic tools
[12, 24, 25, 38]. In order to maximize expression of the cold-active β-galactosidase in Halobacterium sp. NRC-1, we introduced a cold-active promoter for the cold shock protein gene, cspD2, into a haloarchaeal expression vector
. The cspD2 gene was selected based on previous transcriptomic studies of Halobacterium sp. NRC-1
. The combination of high salinity and low temperature induction in NRC-1 led to the successful programmed production of high amounts of active β-galactosidase, nearly 20-fold more than in its natural host.
Another challenge in studies of haloarchaeal proteins has been the development of a purification method, as a result of interference of many analytical and chromatographic techniques by high salinity levels
. For purification of the H. lacusprofundi enzyme overexpressed in Halobacterium sp. NRC-1, we used a combination of ion exclusion chromatography and hydrophobic interaction chromatography, since both methods are distinguished by their ability to be applied at high salinity. It has been observed that proteins with hydrophobic “patches” on their surface tend to bind hydrophobic matrixes, a process that is facilitated by high salt concentrations
[48, 49]. Similarly, ion exclusion chromatography has been successful over a wide range of ionic strength buffers, even those at high salinity
. In the past, ion-exchange chromatography was also used for purification of a mesophilic halophilic β-galactosidase from the haloarchaeon, Haloferax alicantei; however, the temperature profile for this enzyme was not reported
For the H. lacusprofundi β-galactosidase, purity was confirmed by the presence of a highly prominent band on SDS-PAGE, and its identity was verified by LC-MS/MS analysis and enzymatic breakdown of the chromogenic substrates. As previously observed for highly acidic haloarchaeal proteins, anomalous migration was expected during electrophoreses in SDS-PAGE gels, because of the binding of detergents with electrostatic and hydrophobic interactions slows the rate of migration
[41, 51]. Consequently, the bga polypeptide displayed an anomalous molecular mass of ca. 100 kDa, about 28% higher than the predicted molecular mass of 78.06 kDa. However, the protein identity was validated by LC-MS/MS, with 13 peptides covering 14% of the predicted amino acid sequence. The breakdown of the chromogenic substrates, X-gal on agar plates by Halobacterium sp. NRC-1(pMC2) colonies, and ONPG by purified enzyme in solution, confirmed that the β-galactosidase was enzymatically active.
The purified H. lacusprofundi β-galactosidase was found to be extremely halophilic and retained partial activity at cold temperature and surprisingly also at elevated temperature. It exhibited maximal activity in the presence of 4.0 M NaCl/KCl, which are similar to the intracellular ionic composition observed in other haloarchaea
[24, 52]. Halophilic enzymes usually feature an increase in the number of charged amino acids, especially acidic residues at the protein surface and the negative surface charge is critical to their solubility and prevents aggregation at high salt concentrations
[8, 37, 47]. Although the temperature optimum was 50°C for both crude extracts and purified β-galactosidase from Halobacterium sp. NRC-1 (pMC2), the relative enzyme activity at 60°C was slightly higher for the crude extract. A reason for the observed difference could be that the purified enzyme was used without prior addition of stabilizer. The purified β-galactosidase showed a substantial fraction of activity, nearly 13% at 10°C and 10%, at 4°C. Similar temperature characteristics have been previously reported for other cold-active family 42 β-galactosidases from Arthrobacter sp. 32c
 and Carnobacterium sp. BA
, indicating that extremophilic enzymes frequently function suboptimally under physiological conditions. The pH optimum of β-galactosidase was near neutral, similar to other family 42 β-galactosidases
[16, 18, 26] and in contrast to family 2 β-galactosidases, which are optimally active in alkaline conditions
In general, non-halophilic enzymes lose most of their activity in the presence of organic solvents
. Karan et al.
 have recently reported that commercial enzymes lose a significant fraction of activity under similar conditions. The H. lacusprofundi β-galactosidase, in contrast, was found to be remarkably active and stable in aqueous organic solvent mixtures. In previous work, another cold-adapted β-galactosidase from Antarctic bacterium Arthrobacter sp. 32c was also found to be active in similar ethanol concentrations (≤ 20%)
. A protease from halophilic archaeon Natrialba magadii was found to be active and stable in aqueous-organic solvent mixtures containing 1.5 M NaCl and dioxane
. In other studies, halophilic enzymes have been reported to be active and stable in biphasic solutions of water and hydrocarbon organic solvents, such as benzene. These include an amylase of a haloarchaeon, Haloarcula sp. strain S-1
, and a protease from the halophilic bacterium, Geomicrobium sp. EMB2
. These studies indicate that organic solvent stability is a general property of halophilic enzymes, owing to their ability to work at low water activity. However, this is the first report of retention of high levels of enzyme activity in short and long chain alcohols, which reflect the polyextremophilic character of the enzyme.
Polyextremophilic characteristics make the H. lacusprofundi β-galactosidase an ideal candidate for industrial and biotechnological uses. For example, the solvent stability of H. lacusprofundi β-galactosidase can be utilized for synthesis of oligosaccharides in a similar manner to past studies, but with the added benefit of cold activity and halophilicity. Maugard et al.
 have exploited a solvent stable β-galactosidase for the synthesis of galacto-oligosaccharides from lactose. Recently, Bridiau et al.
 reported a tert-butanol stable β-galactosidase from Bacillus circulans that synthesized N-acetyl-lactosamine in hydro-organic media. Vic et al.
 have also reported the synthesis of 2-hydroxybenzyl β-D-glucopyranoside using β-galactosidase in a tert-butanol-water mixture.
The H. lacusprofundi β-galactosidase gene is located in a genomic region encoding proteins for binding, uptake, and catabolism of sugars. Since the environment of Deep Lake does not contain lactose, the β-galactosidase gene and surrounding gene cluster are likely to be involved in degradation and utilization of other carbohydrates, such as plant oligo- and polysaccharides
. These genes reflect a substantial resource for directing the conversion of biomass into valuable commodities, such as biofuels. The properties described for the purified β-galactosidase are likely to be useful for the development and use of haloarchaea in biotechnology. Moreover, our ability to genetically manipulate and shuttle these and other genes represents a substantial resource for the future.
Halophilic Archaea offer an incomparable resource of polyextremozymes which are active and stable in high concentrations of salt, a broad range of temperatures, and organic solvents. At high salinity, water is sequestered in hydrated ionic structures, limiting the availability of free water molecules for protein hydration
. Since halophilic enzymes are adapted to function at high salt concentrations, they are also found to be active at low water availability. Such conditions are also encountered by enzymes in cold temperatures due to the freezing of water molecules and consequent formation of structured ice-like lattices
. Therefore, structure-function studies of cold-active haloarchaeal enzyme, including H. lacusprofundi β-galactosidase, are likely to provide further insights into enzyme catalysis under water limiting conditions, which are likely to enhance their applications in biotechnology.
Since H. lacusprofundi survives in the Antarctic Deep Lake, one of the coldest and most extremely saline environments from which microbes have been cultured, the unusual and unique properties of its enzymes are of general interest to astrobiologists
[5, 9]. Such environments may be analogs of regions of Earth’s sister planet, Mars, where the potential for biological activity is of intense interest. Images from the Mars Reconnaissance Orbiter showed evidence for seasonal emergence of liquid flows in summer, findings consistent with briny liquid water emerging from underground reservoirs on the planet
. The Jovian satellite Europa is covered by frozen water-ice and the presence of liquid water beneath the surface has been hypothesized
[64, 65]. Further studies of model enzymes like β-galactosidase and the polyextremophilic microbe H. lacusprofundi are likely to provide greater insights into how life may be able to cope with challenging conditions on other worlds.