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.
Highlights
Cold habitats are an extraordinary reservoir of biotechnological molecules such as enzymes (Cavicchioli et al, 2011), antimicrobials (Borchert et al, 2017), and biosurfactants (Perfumo et al, 2018) that can function under extreme conditions
As for lipases, bacterial strains were first screened for a general lipolytic activity by placing a volume of 10 μl of a pre-grown culture onto a R2A agar plate supplemented with sunflower oil (1% v/w) and rhodamine B (0.001% v/w) and monitoring under UV irradiation the formation of a fluorescent orange-pink halo around the colony (Beisson et al, 2000)
The 33 bacterial isolates from glacier forefield soils in the Larsemann Hills (East Antarctica) used in this study were largely represented by Actinobacteria (17/33; 52%), of the Arthrobacter genus, followed by Gamma-proteobacteria (7/33; 21%), Alpha-proteobacteria (5/33; 15%), Bacteroidetes (3/33; 9%) and Deinococcus (1/33; 3%) (Figure 1A)
Summary
Cold habitats are an extraordinary reservoir of biotechnological molecules such as enzymes (Cavicchioli et al, 2011), antimicrobials (Borchert et al, 2017), and biosurfactants (Perfumo et al, 2018) that can function under extreme conditions. Cold-adapted enzymes are highly efficient because they can catalyze their reactions with chemical rates comparable to, and often exceeding, those of their warm-active counterparts (Struvay and Feller, 2012; Isaksen et al, 2016) In this context, cold-active enzymes of microbial origin have tremendous biotechnological potential in a large range of markets such as detergents, food and beverages, textiles and can be used for specialty applications in molecular biology/research, pharmaceuticals, and diagnostics. Available coldactive enzymes comprise mostly proteases and lipases, and to lesser extent amylases, cellulases, pectinases, mannanases, and others (Sarmiento et al, 2015) This market is expected to grow further in the coming years pushed forward by the rapid development of new technologies that both lead to the discovery of novel enzymes (e.g., genomics and metagenomics) and enable fine modifications of their chemical composition, structure and functional properties (e.g., genetic engineering and strain optimization or synthetic biology approaches) (Hoesl et al, 2011; Agostini et al, 2017)
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