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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.

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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.
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f3: 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.

Mentions: We investigated the ability of bacterial secretion materials to promote enzyme activity or reactivate heat-shocked enzyme. Four strains of both Gram-negative and Gram-positive bacteria were considered. Culture supernatants of Pseudomonas aeruginosa PAO1, Acinetobacter baumannii AYE, Staphylococcus aureus ATCC 29213 as well as a clinical isolate of Bacillus cereus were investigated for how they affect SsoPox-W263I activity (Fig. 3). Many pathogenic bacteria secrete agents such as proteases or surfactants to inactivate host proteins. Surprisingly, enzyme activity did not decrease in the presence of bacterial supernatant and, in some cases, increased as compared to the LB medium control. Next we considered the ability of supernatants to reactivate heat-shocked enzyme at 150 °C. All the supernatants favored enzyme reactivation, from 40% for P. aeruginosa, up to 80% for B. cereus. To evaluate the thermo-susceptibility of the reactivation phenomenon, supernatants were further heated to precipitate unstable compounds. Interestingly, the reactivation was less pronounced after heating the supernatant for 10 minutes at 100 °C, underlining the fact that thermolabile metabolites and/or proteins are partially responsible for enzyme reactivation.


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

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

f3: 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.
Mentions: We investigated the ability of bacterial secretion materials to promote enzyme activity or reactivate heat-shocked enzyme. Four strains of both Gram-negative and Gram-positive bacteria were considered. Culture supernatants of Pseudomonas aeruginosa PAO1, Acinetobacter baumannii AYE, Staphylococcus aureus ATCC 29213 as well as a clinical isolate of Bacillus cereus were investigated for how they affect SsoPox-W263I activity (Fig. 3). Many pathogenic bacteria secrete agents such as proteases or surfactants to inactivate host proteins. Surprisingly, enzyme activity did not decrease in the presence of bacterial supernatant and, in some cases, increased as compared to the LB medium control. Next we considered the ability of supernatants to reactivate heat-shocked enzyme at 150 °C. All the supernatants favored enzyme reactivation, from 40% for P. aeruginosa, up to 80% for B. cereus. To evaluate the thermo-susceptibility of the reactivation phenomenon, supernatants were further heated to precipitate unstable compounds. Interestingly, the reactivation was less pronounced after heating the supernatant for 10 minutes at 100 °C, underlining the fact that thermolabile metabolites and/or proteins are partially responsible for enzyme reactivation.

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.


Related in: MedlinePlus