Characterization of a novel cold-adapted intracellular serine protease from the extremophile Planococcus halocryophilus Or1

Casper Bøjer Rasmussen; Carsten Scavenius; Ida B Thøgersen; Seandean Lykke Harwood; Øivind Larsen; Gro Elin Kjaereng Bjerga; Peter Stougaard; Jan J Enghild; Mariane Schmidt Thøgersen

doi:10.3389/fmicb.2023.1121857

Characterization of a novel cold-adapted intracellular serine protease from the extremophile Planococcus halocryophilus Or1

Front Microbiol. 2023 Feb 23:14:1121857. doi: 10.3389/fmicb.2023.1121857. eCollection 2023.

Authors

Casper Bøjer Rasmussen¹, Carsten Scavenius², Ida B Thøgersen¹, Seandean Lykke Harwood¹, Øivind Larsen³, Gro Elin Kjaereng Bjerga³, Peter Stougaard⁴, Jan J Enghild¹, Mariane Schmidt Thøgersen⁴

Affiliations

¹ Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark.
² Danish Technological Institute, Aarhus, Denmark.
³ NORCE Climate and Environment, NORCE Norwegian Research Centre AS, Bergen, Norway.
⁴ Department of Environmental Science, Aarhus University, Roskilde, Denmark.

Abstract

The enzymes of microorganisms that live in cold environments must be able to function at ambient temperatures. Cold-adapted enzymes generally have less ordered structures that convey a higher catalytic rate, but at the cost of lower thermodynamic stability. In this study, we characterized P355, a novel intracellular subtilisin protease (ISP) derived from the genome of Planococcus halocryophilus Or1, which is a bacterium metabolically active down to -25°C. P355's stability and activity at varying pH values, temperatures, and salt concentrations, as well as its temperature-dependent kinetics, were determined and compared to an uncharacterized thermophilic ISP (T0099) from Parageobacillus thermoglucosidasius, a previously characterized ISP (T0034) from Planococcus sp. AW02J18, and Subtilisin Carlsberg (SC). The results showed that P355 was the most heat-labile of these enzymes, closely followed by T0034. P355 and T0034 exhibited catalytic constants (k _cat ) that were much higher than those of T0099 and SC. Thus, both P355 and T0034 demonstrate the characteristics of the stability-activity trade-off that has been widely observed in cold-adapted proteases.

Keywords: Planococcus; calcium; characterization; cold adaptation; intracellular subtilisin protease; maturation; protein chemistry.

Grants and funding

This study was supported by the Danish Council for Independent Research (DFF-8022-00385B) and the Novo Nordisk Foundation (BIO-MS) (NNF18OC0032724). GB and ØL acknowledge financial support from the Norwegian Research Council (grant ID: 221568), and basic funding from NORCE Norwegian Research Centre.