Cloning, overexpression, purification, and characterization of a polyextremophilic β-galactosidase from the Antarctic haloarchaeon Halorubrum lacusprofundi

Abstract

Background: Halorubrum lacusprofundi is a cold-adapted halophilic archaeon isolated from Deep Lake, a perennially cold and hypersaline lake in Antarctica. Its genome sequencing project was recently completed, providing access to many genes predicted to encode polyextremophilic enzymes active in both extremely high salinity and cold temperatures.

Results: Analysis of the genome sequence of H. lacusprofundi showed a gene cluster for carbohydrate utilization containing a glycoside hydrolase family 42 β-galactosidase gene, named bga. In order to study the biochemical properties of the β-galactosidase enzyme, the bga gene was PCR amplified, cloned, and expressed in the genetically tractable haloarchaeon Halobacterium sp. NRC-1 under the control of a cold shock protein (cspD2) gene promoter. The recombinant β-galactosidase protein was produced at 20-fold higher levels compared to H. lacusprofundi, purified using gel filtration and hydrophobic interaction chromatography, and identified by SDS-PAGE, LC-MS/MS, and ONPG hydrolysis activity. The purified enzyme was found to be active over a wide temperature range (-5 to 60°C) with an optimum of 50°C, and 10% of its maximum activity at 4°C. The enzyme also exhibited extremely halophilic character, with maximal activity in either 4 M NaCl or KCl. The polyextremophilic β-galactosidase was also stable and active in 10-20% alcohol-aqueous solutions, containing methanol, ethanol, n-butanol, or isoamyl alcohol.

Conclusion: The H. lacusprofundi β-galactosidase is a polyextremophilic enzyme active in high salt concentrations and low and high temperature. The enzyme is also active in aqueous-organic mixed solvents, with potential applications in synthetic chemistry. H. lacuprofundi proteins represent a significant biotechnology resource and for developing insights into enzyme catalysis under water limiting conditions. This study provides a system for better understanding how H. lacusprofundi is successful in a perennially cold, hypersaline environment, with relevance to astrobiology.

Figure 1

H. lacusprofundi carbohydrate utilization gene cluster in chromosome II. The predicted genes, shown to scale, with those in rightward transcriptional orientation in red and leftward orientation in blue. Predicted gene functions are (from left to right) Hlac_2855 and 2857, ISH3 and IS4 family transposases; Hlac_2860, 2-keto-3-deoxy-6-phosphogluconate aldolase/4-hydroxy-2-oxoglutarate aldolase; Hlac_2861, IclR family transcriptional regulator; Hlac_2862, sugar-binding periplasmid protein; Hlac_2863, ABC-type sugar transport system permease component; Hlac_2864, sugar permease; Hlac_2865, ABC-type sugar transport system; ATPase component; Hlac_2866, L-alanine-DL-glutamate epimerase and related enzymes of enolase; Hlac_2867, short-chain dehydrogenase/reductase SDR; Hlac_2868, β-galactosidase; Hlac_2869, α-galactosidase; Hlac_2870, sugar kinase, and Hlac_2871 and 2872, IS200 family and IS605 OrB family transposases.

Figure 2

Shuttle vector pMC2 used for the expression of H. lacusprofundi bga in Halobacterium sp. NRC-1. Location and transcriptional orientation of bla, β-lactamase for ampicillin resistance; mev, HMG-CoA reductase for mevinolin resistance; bga, β-galactosidase, and rep, the Halobacterium pGRB replicase gene are shown. Position of the temperature induced promoter, Pcs→ and KpnI, NdeI and BamHI restriction sites are indicated.

Figure 3

β-galactosidase activity measured in crude extracts of wild-type H. lacusprofundi (blue, ◊) and Halobacterium sp. NRC-1 (pMC2) (red, □) at different temperatures. The enzyme activity in Miller units (MU) for the former haloarchaeon is shown on the left and the latter is shown on the right. Values are the average of triplicate experiments with standard deviation shown with error bars.

Figure 4

Induction of H. lacusprofundi β-galactosidase in Halobacterium sp. NRC-1 (pMC2). Enzyme assays were done at 25°C after incubating cells for 72 hours at various temperatures for cell lysate (blue, ◊) and supernatant (red, □). Activity levels are shown in Miller units (MU) on the left for cell lysate and the right for supernatant, and are the average of triplicate experiments, with standard deviation shown with error bars.

Figure 5

Purification of H. lacusprofundi β-galactosidase overproduced in Halobacterium sp. NRC-1. Proteins are shown electrophoresed on an 8.0% polyacrylamide gel after Coomassie blue staining: Lane 1: Molecular mass markers; Lane 2: Cell lysate; Lane 3: Gel filtration active fractions; Lane 4: HIC active fractions.

Figure 6

Properties of purified H. lacusprofundi β-galactosidase. Effect of various parameters on enzyme activity: (A) varying KCl (blue, ◊), NaCl (red, □) concentrations, measured at 37°C and pH 6.5, (B) varying pH (red, □), measured at 37°C and 4.0 KCl, and varying temperature (blue, ◊), measured at pH 6.5 and 4.0 KCl. Enzyme activity (%) was defined as the percentage of activity detected with respect to the maximum observed β-galactosidase activity in each series. Values are the average of triplicate experiments with standard deviation shown with error bars.

Figure 7

Effect of organic solvents on purified H. lacusprofundi β-galactosidase activity. The enzyme assay was done in presence of 0, 5, or 10% organic solvents. methanol (blue, ◊), ethanol (red, □), n-butanol (black, ∆), isoamyl alcohol (purple, x). Values are the average of triplicate experiments, with standard deviation shown as error bars.