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Harnessing hyperthermostable lactonase from Sulfolobus solfataricus for biotechnological applications

View Article: PubMed Central - PubMed

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.

No MeSH data available.


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.
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f4: 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.

Mentions: Hyperthermostable enzymes are commonly described as nearly inactive at room temperature, but their catalytic power is in fact greater at lower temperature34950. Optimum SsoPox-W263I activity was previously determined in the range of 80–95 °C and significant activity was also observed at 23 °C143048. Here, we investigated the ability of the enzyme to work at sub-zero in glycerol-complemented buffer (Fig. 4). Surprisingly, the variant was still significantly active at −18 °C. The kcat/KM values were estimated using a low ethyl paraoxon concentration (250 μM) and values of 2.0 × 104 M−1.s−1, 9.7 × 103 M−1.s−1 and 1.4 × 103 M−1.s−1 were determined at 70 °C, 23 °C and −18 °C respectively (Table 1). Rates only decreased by 14-fold and 7-fold at −18 °C, as compared to 70 °C and room temperature, and converted more than 99% of substrate in 6 hours with only 0.3 μM of enzyme. In a previous work, the temperature coefficient (Q10) for > 100 enzymatic reactions was found to be 2, including those involving thermostable enzymes49. Thus, the average enzymatic rate decrease by ~446-fold and ~17-fold over the same temperature ranges, 70 °C/−18 °C and 23 °C/−18 °C, respectively. The temperature dependence of SsoPox-W263I is therefore much lower than the average enzyme. Consequently, the temperature coefficient values calculated between 70 °C/23 °C, 70 °C/−18 °C and 23 °C/−18 °C varying from 1.17 to 1.60 were found to be significantly lower than for most enzymes ( value is ≈2)49.


Harnessing hyperthermostable lactonase from Sulfolobus solfataricus for biotechnological applications
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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5120315&req=5

f4: 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.
Mentions: Hyperthermostable enzymes are commonly described as nearly inactive at room temperature, but their catalytic power is in fact greater at lower temperature34950. Optimum SsoPox-W263I activity was previously determined in the range of 80–95 °C and significant activity was also observed at 23 °C143048. Here, we investigated the ability of the enzyme to work at sub-zero in glycerol-complemented buffer (Fig. 4). Surprisingly, the variant was still significantly active at −18 °C. The kcat/KM values were estimated using a low ethyl paraoxon concentration (250 μM) and values of 2.0 × 104 M−1.s−1, 9.7 × 103 M−1.s−1 and 1.4 × 103 M−1.s−1 were determined at 70 °C, 23 °C and −18 °C respectively (Table 1). Rates only decreased by 14-fold and 7-fold at −18 °C, as compared to 70 °C and room temperature, and converted more than 99% of substrate in 6 hours with only 0.3 μM of enzyme. In a previous work, the temperature coefficient (Q10) for > 100 enzymatic reactions was found to be 2, including those involving thermostable enzymes49. Thus, the average enzymatic rate decrease by ~446-fold and ~17-fold over the same temperature ranges, 70 °C/−18 °C and 23 °C/−18 °C, respectively. The temperature dependence of SsoPox-W263I is therefore much lower than the average enzyme. Consequently, the temperature coefficient values calculated between 70 °C/23 °C, 70 °C/−18 °C and 23 °C/−18 °C varying from 1.17 to 1.60 were found to be significantly lower than for most enzymes ( value is ≈2)49.

View Article: PubMed Central - PubMed

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.

No MeSH data available.