Discovery and Characterization of a New Cold-Active Protease From an Extremophilic Bacterium via Comparative Genome Analysis and in vitro Expression

Amedea Perfumo; Georg Johannes Freiherr von Sass; Eva-Lena Nordmann; Nediljko Budisa; Dirk Wagner

doi:10.3389/fmicb.2020.00881

Discovery and Characterization of a New Cold-Active Protease From an Extremophilic Bacterium via Comparative Genome Analysis and in vitro Expression

Front Microbiol. 2020 May 13:11:881. doi: 10.3389/fmicb.2020.00881. eCollection 2020.

Authors

Amedea Perfumo^{1

2}, Georg Johannes Freiherr von Sass³, Eva-Lena Nordmann^{1

4}, Nediljko Budisa^{3

5}, Dirk Wagner^{1

6}

Affiliations

¹ GFZ German Research Centre for Geosciences, Helmholtz Centre Potsdam, Section Geomicrobiology, Potsdam, Germany.
² Polar Terrestrial Environmental System Division, Helmholtz Centre for Polar and Marine Research, Alfred Wegener Institute, Potsdam, Germany.
³ Institute of Chemistry, Technische Universität Berlin, Berlin, Germany.
⁴ Institute of Chemistry and Biology of the Marine Environment, Carl-von-Ossietzky Universität Oldenburg, Oldenburg, Germany.
⁵ Institute of Chemistry, University of Manitoba, Winnipeg, MB, Canada.
⁶ Institute of Geosciences, University of Potsdam, Potsdam, Germany.

Abstract

Following a screening of Antarctic glacier forefield-bacteria for novel cold-active enzymes, a psychrophilic strain Psychrobacter sp. 94-6PB was selected for further characterization of enzymatic activities. The strain produced lipases and proteases in the temperature range of 4-18°C. The coding sequence of an extracellular serine-protease was then identified via comparative analysis across Psychrobacter sp. genomes, PCR-amplified in our strain 94-6PB and expressed in the heterologous host E. coli. The purified enzyme (80 kDa) resulted to be a cold-active alkaline protease, performing best at temperatures of 20-30°C and pH 7-9. It was stable in presence of common inhibitors [β-mercaptoethanol (β-ME), dithiothreitol (DTT), urea, phenylmethylsulfonyl fluoride (PMSF) and ethylenediaminetetraacetic acid (EDTA)] and compatible with detergents and surfactants (Tween 20, Tween 80, hydrogen peroxide and Triton X-100). Because of these properties, the P94-6PB protease may be suitable for use in a new generation of laundry products for cold washing. Furthermore, we assessed the microdiversity of this enzyme in Psychrobacter organisms from different cold habitats and found several gene clusters that correlated with specific ecological niches. We then discussed the role of habitat specialization in shaping the biodiversity of proteins and enzymes and anticipate far-reaching implications for the search of novel variants of biotechnological products.

Keywords: bioprospecting; cold-active enzymes; extremophilic bacteria; genome mining; heterologous protein expression; microdiversity.