Red Sea Atlantis II brine pool nitrilase with unique thermostability profile and heavy metal tolerance

Abstract

Background: Nitrilases, which hydrolyze nitriles in a one-step reaction into carboxylic acids and ammonia, gained increasing attention because of the abundance of nitrile compounds in nature and their use in fine chemicals and pharmaceutics. Extreme environments are potential habitats for the isolation and characterization of extremozymes including nitrilases with unique resistant properties. The Red Sea brine pools are characterized by multitude of extreme conditions. The Lower Convective Layer (LCL) of the Atlantis II Deep Brine Pool in the Red Sea is characterized by elevated temperature (68 °C), high salt concentrations (250 ‰), anoxic conditions and high heavy metal concentrations.

Results: We identified and isolated a nitrilase from the Atlantis II Deep Brine Pool in the Red Sea LCL. The isolated 338 amino-acid nitrilase (NitraS-ATII) is part of a highly conserved operon in different bacterial phyla with indiscernible function. The enzyme was cloned, expressed and purified. Characterization of the purified NitraS-ATII revealed its selectivity towards dinitriles, which suggests a possible industrial application in the synthesis of cyanocarboxylic acids. Moreover, NitraS-ATII showed higher thermal stability compared to a closely related nitrilase, in addition to its observed tolerance towards high concentrations of selected heavy metals.

Conclusion: This enzyme sheds light on evolution of microbes in the Atlantis II Deep LCL to adapt to the diverse extreme environment and can prove to be valuable in bioremediation processes.

Fig. 1

The NitraS-ATII protein. a Amino acid sequence of the native NitraS-ATII with the catalytic triad of glutamate (D47), lysine (K129) and cysteine (C163) shown in red. b Three dimensional model of NitraS-ATII protein. The model was obtained using Phyre2 tool with 100 % confidence and visualized using pyMOL. Secondary structures were colored as follows: α-helices in red, β-sheets in yellow and loops in green. Residues of the catalytic triad are shown as stick representation, with carbon atoms in cyan, nitrogen atoms in red, oxygen atoms in blue and sulfur atoms in yellow. c Superimposition of three-dimensional structure models of NitraS-ATII and nitrilase from Rhodobacter sphaeroides LHS-305. NitraS-ATII, colored according to secondary structure, was superimposed onto R. sphaeroides LHS-305 nitrilase, shown in blue. Residues of the catalytic triad showed perfect superimposition

Fig. 2

Purification of NitraS-ATII protein. Protein concentration and integrity at different purification steps were visualized on SDS-PAGE gel (12 %). Lane 1: ProSieve® Colour Protein Markers (Lonza), lane 2: non-transformed cells supernatant, lane 3: uninduced cells supernatant, lane 4: induced supernatant before purification, lane 5: NTA column flow-through, lane 6: first column wash, lane7: second column wash, lane 8: eluate fraction one, lane 9: eluate fraction two, lane 10: eluate fraction three

Fig. 3

Biochemical characterization of NitraS-ATII. a. Effect of pH on nitrilase activity was assessed in acetate buffer (pH 3.5–5.0), phosphate buffer (pH 6.0–8.0) and carbonate buffer (pH 9.0–11.0). Optimum activity was achieved at pH 7.0. b. Effect of temperature on nitrilase activity. Optimum temperature was determined to be 40 °C and activity was nearly abolished at higher temperatures. c. Residual nitrilase activity after incubation at different temperatures for different periods. d. Nitrilase activity at different NaCl concentrations. Decrease in activity was observed with increase in salt concentration. All assays used 400 mmol.L^-1 succinonitrile as substrate

Fig. 4

Thermal stability of NitraS-ATII. The enzymes were incubated at high temperatures for 30 s (a) or 1 min (b) prior to performing the reaction and measuring the residual activity. Two-way ANOVA test followed by Bonferroni post-hoc test was performed using GraphPad Prism®(Windows® version 5.00). *** and ** indicate p-values lower than 0.001 and 0.01, respectively

Fig. 5

Effect of succinonitrile concentration on NitraS-ATII specific activity. Specific activity was measured in U. One unit (U) of specific activity is defined as 1.0 micromole of ammonia produced in 1.0 min by the enzyme (μmol.min^-1) under used reaction conditions

Fig. 6

Effect of different metal ions on NitraS-ATII activity. The nitrilase activity is retained at high concentrations of Mg²⁺ and Mn²⁺ and to a lower extent with Zn²⁺. A high degree of tolerance is also observed towards Cd²⁺and Co²⁺, and to a lower extent towards Ni²⁺. Inactivation is observed even with low concentrations of Cu²⁺ and Hg²⁺

Fig. 7

Comparison of the effect of selected metal ions on the activities of NitraS-ATII and R. sphaeriodesLHS-305 nitrilase. Each panel shows the activity percentage in the presence of increasing concentrations of a metal ion. a. In presence of Ni²⁺, R. sphaeriodes LHS-305 nitrilase retains higher activity (t test p-value = 2.2x10^-3). b. In presence of Zn²⁺, NitraS-ATII retains higher activity (t test p-value = 9.6x10^-3). c. In presence of Mn²⁺, NitraS-ATII retains higher activity (t test p-value = 11.8x10^-3)