Abstract
Heterologous production of proteins in Escherichia coli has raised several challenges including soluble production of target proteins, high levels of expression and purification. Fusion tags can serve as the important tools to overcome these challenges. SUMO (small ubiquitin-related modifier) is one of these tags whose fusion to native protein sequence can enhance its solubility and stability. In current research, a simple, efficient and cost-effective method is being discussed for the construction of pET28a-SUMO vector. In order to improve the stability and activity of lysophospholipase from Pyrococcus abyssi (Pa-LPL), a 6xHis-SUMO tag was fused to N-terminal of Pa-LPL by using pET28a-SUMO vector. Recombinant SUMO-fused enzyme (6 H-S-PaLPL) works optimally at 35 °C and pH 6.5 with remarkable thermostability at 35–95 °C. Thermo-inactivation kinetics of 6 H-S-PaLPL were also studied at 35–95 °C with first order rate constant (kIN) of 5.58 × 10− 2 h-1 and half-life of 12 ± 0 h at 95 °C. Km and Vmax for the hydrolysis of 4-nitrophenyl butyrate were calculated to be 2 ± 0.015 mM and 3882 ± 22.368 U/mg, respectively. 2.4-fold increase in Vmax of Pa-LPL was observed after fusion of 6xHis-SUMO tag to its N-terminal. It is the first report on the utilization of SUMO fusion tag to enhance the overall stability and activity of Pa-LPL. Fusion of 6xHis-SUMO tag not only aided in the purification process but also played a crucial role in increasing the thermostability and activity of the enzyme. SUMO-fused enzyme, thus generated, can serve as an important candidate for degumming of vegetable oils at industrial scale.
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The data used in the current research work will be provided by the corresponding author upon reasonable request.
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Acknowledgements
We acknowledge School of Biological Sciences, University of the Punjab, Lahore (Pakistan) for providing research facilities.
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The entire research work was supported by School of Biological Sciences, University of the Punjab, Lahore (Pakistan).
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All the authors contributed to the study conception and design. All the authors read and approved the final version of the manuscript. Conceptualization, investigation, methodology, data curation, formal analysis and writing-original draft were performed by Arshia Nazir. Task of data curation and methodology was completed by Mohsin Shad. Hafiz Muzzammel Rehman has completed in silico structural and functional analysis of SUMO-fused enzyme. Data curation and reviewing were completed by Naseema Azim. Conceptualization, fund acquisition, supervision, project administration, reviewing and writing final draft were done by Muhammad Sajjad*.
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The authors (Arshia Nazir, Mohsin Shad, Hafiz Muzzammel Rehman, Naseema Azim and Muhammad Sajjad*) declare that they have no known competing or financial interests or personal relationships that could have appeared to influence the research work reported in this paper.
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Nazir, A., Shad, M., Rehman, H.M. et al. Application of SUMO fusion technology for the enhancement of stability and activity of lysophospholipase from Pyrococcus abyssi. World J Microbiol Biotechnol 40, 183 (2024). https://doi.org/10.1007/s11274-024-03998-w
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DOI: https://doi.org/10.1007/s11274-024-03998-w