Harnessing hyperthermostable lactonase from Sulfolobus solfataricus for biotechnological applications

Abstract

Extremozymes have gained considerable interest as they could meet industrial requirements. Among these, SsoPox is a hyperthermostable enzyme isolated from the archaeon Sulfolobus solfataricus. This enzyme is a lactonase catalyzing the hydrolysis of acyl-homoserine lactones; these molecules are involved in Gram-negative bacterial communication referred to as quorum sensing. SsoPox exhibits promiscuous phosphotriesterase activity for the degradation of organophosphorous chemicals including insecticides and chemical warfare agents. Owing to its bi-functional catalytic abilities as well as its intrinsic stability, SsoPox is appealing for many applications, having potential uses in the agriculture, defense, food and health industries. Here we investigate the biotechnological properties of the mutant SsoPox-W263I, a variant with increased lactonase and phosphotriesterase activities. We tested enzyme resistance against diverse process-like and operating conditions such as heat resistance, contact with organic solvents, sterilization, storage and immobilization. Bacterial secreted materials from both Gram-negative and positive bacteria were harmless on SsoPox-W263I activity and could reactivate heat-inactivated enzyme. SsoPox showed resistance to harsh conditions demonstrating that it is an extremely attractive enzyme for many applications. Finally, the potential of SsoPox-W263I to be active at subzero temperature is highlighted and discussed in regards to the common idea that hyperthermophile enzymes are nearly inactive at low temperatures.

Figure 1. Relative activity of SsoPox-W263I submitted to a 5-minute heat shock in either liquid (blue) or solid form (brown).

The values represent the mean ± SEM (Standard Error to the Mean) of six replicates.

Figure 2

SsoPox-W263I relative activity after submission to three sterilization methods: autoclave (a), ethylene oxide (b), β-radiation (c). Experiments on solid enzyme are in brown bars while those on liquid enzyme are in blue. Measures were performed in triplicate as compared to a non-sterilized control. Values represent means ± SEM.

Figure 3

Dot plots of the relative activity of SsoPox-W263I in contact with bacterial culture supernatants of P. aeruginosa PAO1 (a), A. baumannii AYE (b), B. cereus (c) and S. aureus (d). For each graph, blue dots correspond to the positive control (i.e. unheated enzyme resuspended in sterile LB medium). Red dots correspond to the heated control (i.e. enzyme heated at 150 °C and resuspended in sterile LB medium). Green dots represent the enhanced activity of unheated enzyme resuspended in bacterial supernatant. Orange dots illustrate the reactivation of heat-shocked enzyme resuspended in bacterial supernatant. Finally, purple dots represent the activity of heated enzyme resuspended in previously 100 °C heated bacterial supernatant to investigate the role of thermolabile compounds in the reactivation phenomenon. The n = 6 replicates are represented with mean ± SEM (Standard Error to the Mean) in black bars. Black stars (*) indicate a significant difference (p < 0.01) according to the corresponding contrast.

Figure 4. Hydrolysis of paraoxon by variant SsoPox-W263I over time at 70 °C (red dots), 23 °C (orange dots) and −18 °C (blue dots) with 50% glycerol.

Relative concentrations of paraoxon are presented. Measures were performed in triplicate and are represented with mean ± SEM in black bars.

Figure 5. Relative activities of SsoPox-W263I after treatments in different solvents.

For each solvent, the n = 6 replicates are represented with mean ± SEM in black bars.

Figure 6. Dot plots of the relative activity of solid enzyme stored at room temperature over time.

For each time, the n = 3 replicates are represented with mean ± SEM in black bars.

Figure 7. Immobilization rate of enzyme by crosslinking in alginate beads as compared to the free enzyme and recovery.

The results for each cycle are represented with mean ± SEM in black bars. Immobilized and released activities are shown in brown and grey respectively.